Prostatic membranes contain high affinity [dissociation constant (KD) = 1.16 nM], saturable binding sites for [125I]iodo-epidermal growth factor (EGF). The binding of [125I]iodo-EGF is specific since it is displaced by excess EGF but not by insulin, fibroblast growth factor, platelet-derived growth factor, or multiplication-stimulating activity. Affinity labeling with [125I]iodo-EGF and subsequent cross-linking with disuccinimidyl suberate demonstrated the specific binding of [125I]iodo-EGF to a macromolecule with a mol wt of 170,000. Castration of mature rats resulted in a 3- to 6-fold increase in [125I]iodo-EGF binding, while treatment of 7-day castrated rats with 5 alpha-dihydrotestosterone decreased the number of binding sites. Administration of estrogen or progesterone produced a slight decrease in EGF binding sites but not nearly to the extent observed with 5 alpha-dihydrotestosterone, suggesting that the observed effect is androgen specific. These results demonstrate that rat prostate contains specific binding sites for EGF and that their level is modulated by androgens.