Mammary explants from virgin and pregnant Balb/c mice were incubated with 3H-thymidine, and its incorporation into TCA-precipitable material was determined. In addition, the labeling index of alveolar and ductal epithelial cells was determined by autoradiography. Incorporation of precursor into mammary tissue from pregnant animals was elevated above that of virgin animals on each day of pregnancy. During pregnancy, there were three periods of peak incorporation (on days 3, 7, and 11-15). The labeling index of epithelial cells also increased during this period. Ductal cells had a peak labeling index on day 3 (30%), which dropped thereafter to comparatively low levels (3-6%) for the rest of pregnancy. With the exception of days 1 and 3, the labeling index of ductal cells showed no difference between midnight and noon. Alveolar structures were first observed in histological preparations on day 5 of pregnancy. The labeling index of alveolar cells was higher than that of ductal cells during the rest of pregnancy, and on most days showed a diurnal fluctuation, being greater at midnight than at noon. The labeling index of alveolar cells had peak values at midnight on days 7 (23%) and 11-13 (20%). This daily fluctuation in DNA synthesis by alveolar cells is a circadian rhythm; pregnant animals continued to show this rhythm after 13 days in constant darkness, and animals maintained on a reversed light schedule had a reversed rhythm. Mammary tissue shows a complex pattern of DNA synthesis during pregnancy, reflecting the different characteristics of ductal and alveolar epithelial cells.