Recent observations indicate that the progesterone metabolite, 5 alpha-pregnane-3,20-dione (5 alpha P), which is produced at higher levels in, tumorous breast tissue, promotes cell proliferation and detachment, whereas 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), which is produced at higher levels in nontumorous breast tissue, suppresses proliferation and detachment of MCF-7 breast cancer cells. The objective of the current study was to determine the presence and characteristics of binding sites for these endogenous putative cancer-regulating steroid hormones. Radiolabeled 5 alpha P and 3 alpha HP were used in radioligand binding assays on MCF-7 cell (membrane, cytosolic, and nuclear) fractions. Binding of [H-3]5 alpha P and [H-3]3 alpha HP was observed only in the plasma membrane fraction, whereas estradiol binding sites were confirmed in the cytosolic and nuclear fractions. The respective membrane binding sites exhibited specificity for the 5 alpha P and 3 alpha HP ligands with no appreciable displacement at 200- to 500-fold excess by other steroids. The association rate constants were calculated as 0.107/min and 0.0089/min and the dissociation rate constants were 0.049 9 and 0.011 for 5 alpha P and 3 alpha HP, respectively. Saturation analyses indicated single classes of molecules with dissociation constants of 4.5 and 4.87 nM and receptor densities of 486 and 629 fmol/mg protein, respectively, for 5 alpha P and 3 alpha HP. Exposure of MCF-7 cells to estradiol for 1, 24, 48, and 72 h resulted in 2.3, 4.2-, 2.99-, and 1.7-fold increases, respectively, in 5 alpha P receptor density. 3 alpha HP resulted in partial suppression of the estradiol mediated increase in 5 alpha P receptor density. This is the first report of receptors for the progesterone metabolites, 5 alpha P and 3 alpha HP, of their occurrence in breast cancer cell membranes, and of the induction of 5 alpha P receptors by estradiol. The results provide further support for the potential importance of progesterone metabolites in breast cancer.