DNA synthesis in mammary gland estimated by [3H]thymidine incorporation was significantly higher on the day of proestrus than on the 2nd day of diestrus in 50 day old female Sprague-Dawley rats. The percentage of progressive mammary tumors, tumor growth rate and the number and weight of tumors per tumor-bearing rat were significantly higher in animals given a single i.v. injection of 5 mg 7,12-dimethylbenz(a)anthracene at proestrus than in the animals given it at diestrus. Inhibition of DNA synthesis at proestrus by 2-bromo-.alpha.-ergocryptine also suppressed mammary tumorigenesis by the carcinogen. In 90 day old rats in which little difference was found in mammary gland DNA synthesis between proestrus and diestrus, there was no difference in mammary tumorigenesis between animals given the carcinogen at proestrus and animals given it at diestrus. Prestimulation of mammary gland DNA synthesis by prolactin increased the growth, number and weight of carcinogen-induced mammary tumors. These results demonstrate the importance of mammary DNA synthesis at the time when a carcinogen acts on the glands in mammary tumorigenesis.