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Hexadecanoate was translocated in Nocardia asteroides by a constitutive transport system(s), which transported short, medium, and long-chain fatty acids. Inhibition of hexadenocanoate transport by homologues suggested that at least two systems are present: one specific for short-chain fatty acids and the other specific for medium- and long-chain fatty acids. Saturation kinetics typical of a carrier-mediated transport system (Kt = 870 muM)were observed, and concentration of fatty acids against a gradient was achieved. Inhibitor studies indicated that free sulfhydryl groups, a functional respiratory chain, and energy are required for translocation. Efflux of [14C]hexadecanoate in the presence of excess unlabeled hexadecanoate or 2,4-dinitrophenol and the cytoplasmic localization of acyl-coenzyme A synthetase (acid:coenzyme A ligase [adenosine monophosphate]; EC 6.2.1.3) (Calmes and Deal, 1973) are consistent with the hypothesis that fatty acids are transported and released intracellularly as free fatty acids.