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Chronic treatment of rats with the synthetic estrogen diethylstilbestrol is known to induce the formation of pituitary tumors, and such tumor induction is highly dependent on the strain of rat used. We examined three previously discovered restriction fragment length polymorphisms in rats to determine whether these correlated with susceptibility to tumor formation. The results indicate that the presence of particular alleles of the polymorphic N-ras and retinoblastoma (Rb) genes does not correlate with tumor susceptibility. A polymorphism upstream of the rat prolactin (Prl) gene is due to the presence or absence of an Alu-like sequence. Results of this study indicate that animals bearing the allele lacking this Alu-like insertion are more likely to develop larger pituitary tumors in response to diethylstilbestrol than are animals in which the Prl allele contains the insertion. In addition, we show that the N-ras, Rb, and Prl genes are dispersed in the rat genome and that the polymorphic alleles of the Prl genes are segregating as classical Mendelian alleles. These results suggest that the difference in the Prl gene itself or in some closely linked gene is related to tumor resistance or susceptibility.