Women who bear their first child by their late teens have about half the risk of developing breast cancer relative to nulliparous women. The rat is a good model for studying the role of hormones in breast cancer since, for example, young rats become nearly refractory to mammary carcinogenesis after delivering a litter of pups. Short term administration of estradiol and progesterone (E and P) provides virgin rats protection from mammary carcinogenesis as effectively as pregnancy. The purpose of these studies were twofold: first, to evaluate potential long-term toxicity of the E and P treatments and second, to compare hormone treated rats and pregnant rats with respect to circulating E and P levels as well as mammary epithelial cell proliferation and differentiation. To test for toxicity, rats were treated with E and P (20 μg and 4 mg, respectively) or vehicle by s.c. injections 5 times per week for 5 weeks beginning at 40 days of age. The animals were weighed biweekly and sacrificed at 500 days of age when detailed necropsies were performed. No significant difference in weight gain was observed between the two groups nor was any toxicity grossly observable in the hormone-treated rats. Furthermore, there was no increase in the number of spontaneous mammary or pituitary tumors in the E and P treated group relative to controls. To evaluate serum hormone titers and mammary proliferation, rats were treated with steroids or vehicle daily beginning at 65 days of age. At 6 and 24 hours after the 1st, 14th and 35th injection, serum E and P were measured by RIA and mammary epithelial cell proliferation by immunohistochemistry (PCNA). At 6 hours after each injection, E and P levels were 3 to 5 fold those observed late in pregnancy. By 24 hours, however, E and P levels subsided to late pregnancy levels or lower. The mammary epithelial cell proliferation index in either E and P treated or late pregnant rats was 6 to 14%. Histologic sections and wholemounts of mammary glands showed a similar degree of differentiation between rats treated with E and P for 14 days or longer and late pregnant rats. These data further suggest that E and P treatments are a non-toxic means of mimicking the protective effect of pregnancy against mammary cancer and that pregnancy or hormone treatments may achieve this prophylaxis through a differentiation mechanism.