Prolactin reverses the inhibitory effects of pharmacological doses of androgen on 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth (Quadri, S.K., Kledzik, G.S., and Meites, J.J. Natl. Cancer Inst., 52: 875-878,1974). To determine whether this effect is due to an alteration in the ability of the tumor cell to bind prolactin, we have quantitated prolactin receptors in androgen-responsive and nonresponsive tumors. Prolactin receptors were measured with 125I-labeled ovine prolactin in a subcellular fraction which reproducibly contained 60 to 80% of the total receptor present in tumor homogenates. Prolactin binding was reversible, reached a steady state in 9 hr, and was completed by excess unlabeled prolactin. Prolactin bound to its receptor with a Kd of approximately 1 X 10(-10) M. Growing tumors were biopsied, and rats bearing regrown tumors were given injections of 4 mg testosterone propionate twice a week. Prolactin receptors were reduced in most of the tumors, which regressed after testosterone treatment by an average of 63% compared to the pretreatment biopsy specimens. Nonresponsive tumors and vehicle-injected controls showed no signifcant alterations in receptor content. This reduction of prolactin receptors is probably insufficient to account for androgen-induced mammary tumor regression.