Recently, several investigators have demonstrated the presence of triphenylethylene antiestrogen binding sites in the cytoplasm of many tissues, which specifically bind to radioactive tamoxifen with high affinity. Although mammary gland is one of the principal target organs for antiestrogen action, the characterization of antiestrogen binding in mammary tissues has not been reported. We have studied the antiestrogen binding properties of [3H]tamoxifen in the mammary glands of virgin, pregnant and lactating rats as well as in the N-methyl-N-nitrosourea-induced mammary tumors. Tritium labeled tamoxifen bound specifically and with high affinity (Kd = 10––9M) to components present in 25,000 g supernatant. Unlabeled estradiol or DES did not compete for these sites, whereas unlabeled tamoxifen showed competitive inhibition. The mammary glands contained threefold higher levels of cytoplasmic binding sites as compared to the mammary tumors. Mammary glands from the pregnant rats bound tamoxifen to a greater extent than that of either virgin or lactating rats. The functional relevance of these binding sites is still unknown.