To investigate the role of hormones in regulating growth of neoplastic mammary cells, we established a heterologous assay for studying interactions of partially purified calf uterine [3H]estradiol-charged estrogen receptor ([3H]ER) with rat tumor nuclei in vitro. This system displays saturable high affinity binding of [3H]ER which is time and salt dependent. Optimal assay conditions required for the heterologous system were identical to those we reported for the homologous calf nuclear binding system. Specificity of [3H]ER binding was demonstrated; 10-fold excess unlabeled estrogen-charged ER (EcR) competed for greater than 90% of the [3H]ER binding sites and binding of [3H]estradiol (not complexed with ER) was less than 1% of [3H]ER binding. Binding of [3H]ER displayed tissue specificity in decreasing order: R3230AC mammary tumor greater than lactating mammary gland = liver greater than kidney greater than lung. Scatchard analysis of saturation data provided estimates of binding affinity to nuclei from R3230AC mammary tumors [Kd, 2.0 +/- 0.3 (SE) nM); the number of binding sites per nucleus for R3230AC tumors was 95,000 +/- 13,800. [3H]ER binding to nuclei isolated from R3230AC rat mammary tumors grown in intact rats was 40% higher than that observed in tumors from ovariectomized animals. Results of administration of individual pharmacological doses of either progesterone or an estrogen to ovariectomized rats did not restore nuclear ER binding levels in R3230AC tumors to those detected in tumors from intact rats. These results suggest that the physiological levels of endogenous hormones produced by the ovaries are important in regulating the number of ER binding sites in nuclei from these mammary tumors.