Androgens are key regulators of male sexual differentiation during the in utero and early postnatal development. Exposure to chemicals that counteract androgen action at some stage in this period can permanently demasculinize male fetuses and lead to malformations of the reproductive tract. Examples of chemicals known to disrupt sexual differentiation in this way include pesticides and their metabolites, such as vinclozolin, procymidone, 1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene (p,p′-DDE), and linuron, and certain phthalate esters such as di-ethylhexyl phthalate and di-butyl phthalate (Gray et al. 2000, 2001). Reduced anogenital distance, retention of nipples or areolas, hypospadias, agenesis of sex accessory tissues, and undescended testes have been described as consequences of disruption of androgen action in the developing rat. These effects are thought to arise through antagonism of androgens at the steroid receptor level and/or via suppression of testosterone synthesis in Leydig cells (Fisher 2004; Gray et al. 2001). Many anti-androgenic chemicals have been found as mixtures in humans (Blount et al. 2000; Swan et al. 2005), including children (Brock et al. 2002; Main et al. 2006), and in wildlife (Guillette 2000). These findings have stimulated interest in exploring the consequences of combined exposures to anti-androgens, although relatively few studies have addressed the issue. There is good evidence that inhibition of androgen binding and other receptor-mediated events occur in an additive fashion (Birkhoj et al. 2004; Gray et al. 2001; Nellemann et al. 2003), but little is known about the developmental effects of in utero and early postnatal exposure to multiple anti-androgenic chemicals. In this article, we present data from detailed investigations of the ability of combinations of androgen receptor (AR) antagonists to induce disruption of male sexual differentiation after long-term exposures in utero and postnatally. We selected a mixture of vinclozolin, procymidone, and flutamide for our experiments. Vinclozolin metabolites compete with androgens for AR binding (Kelce et al. 1994), suppress androgen-dependent gene transcription (Kelce et al. 1997), and affect reproductive development. Procymidone and flutamide also antagonize competitively the AR binding of androgens, with consequent inhibition of AR-mediated gene expression (Ostby et al. 1999; Simard et al. 1986). Common developmental effects of all three chemicals after in utero exposure of male rats include reduced anogenital distance (AGD), nipple retention (NR), hypospadia, diminished prostate weight, reduced testis and epididymal weights, and altered behavior in male offspring (Foster and McIntyre 2002; Gray et al. 1994; Hellwig et al. 2000; Hib and Ponzio 1995; Hotchkiss et al. 2002; McIntyre et al. 2001; Miyata et al. 2002; Ostby et al. 1999; Shimamura et al. 2002). There is no particular environmental relevance to this mixture. The choice of compounds was motivated by our interest to explore the predictability of combination effects caused by similarly acting anti-androgens rather than to emulate “real world” mixtures. Conclusive answers to the question of combination effect predictability require quantitative comparisons between predicted and experimentally observed mixture effects. Experimentally, we have approached this task in a step-wise fashion: a) Dose–response curves for all single-mixture components were recorded. b) These data were used for the calculation of additivity expectations for a mixture of specific composition using “fixed mixture ratio design” (Altenburger et al. 2000; Hewlett and Plackett 1959). c) The mixture experiments were conducted. d) The observed combination effects were compared with the predicted responses. The choice of an appropriate model for the calculation of additivity expectations is essential for assessments of mixture effects because it is in relation to these additivity expectations that combination effects are judged in terms of synergisms or antagonisms. Several concepts for the computation of expected additive effects of anti-androgens have been used. The simple method of summing the individual effects of chemicals in the combination, termed “effect summation,” has been drawn on previously (Gray et al. 2001) but produces unreliable results with sigmoidal dose–response curves (Kortenkamp and Altenburger 1998). The concept of dose addition, also referred to as “concentration addition” (Loewe and Muischnek 1926), is usually employed for combinations of chemicals with similar modes of action. It has previously given additivity expectations well in agreement with experimental observations for inhibition of AR binding and AR-mediated responses in vitro and in vivo (Birkhoj et al. 2004; Nellemann et al. 2003). In light of these observations, we reasoned that dose addition would also produce valid additivity expectations for developmental effects after prolonged in utero and postnatal exposures. Although a series of articles have been published describing the successful application of the fixed mixture ratio approach to in vitro systems (Altenburger et al. 2000; Backhaus et al. 2000; Payne et al. 2001; Rajapakse et al. 2002, 2004; Silva et al. 2002), there is comparatively little experience with in vivo assays. In the endocrine disruptor field, Brian et al. (2005) have recently demonstrated the usefulness of this method to the assessment of multicomponent mixtures of estrogenic chemicals in fish, but there are as yet no examples with mammalian assays in vivo. Thus, to make the assessment of developmental effects of mixtures of chemicals a viable proposition, a number of practical requirements had to be considered. Of particular importance were demands of minimal data variation and high reproducibility. When dealing with several mixture components and a large number of dose levels, the parallel testing of all agents and their mixtures is not a realistic option, especially not with in vivo experiments. Thus, reliance had to be made on historical data, in some cases recorded more than a year before commencement of the mixture experiments, and this placed great emphasis on the reproducibility of test outcomes. We considered that the high demands in terms of data variation were more likely to be met with developmental end points that lend themselves to straight-forward quantification. For these reasons, we selected changes in AGD and NR in male offspring of rats as main end points for our mixture experiments. Both these end points are sensitive to anti-androgen exposure. The aim of our studies was to assess whether the joint effects of mixtures of AR antagonists can be predicted accurately over a large effect range on the basis of dose–response data of the individual components. We reasoned that if there are demonstrable consistent relationships between the potency of individual chemicals and the ways in which they act together, powerful tools for prospective risk assessment would become available. These tools could open the way to make productive use of existing single-chemical databases for the prediction of mixture effects. A second aim was to determine whether there would be joint effects when every mixture component was present at doses that individually do not produce observable responses.