The effect of pregnancy on postweaning mammary gland involution was investigated in mice. On the third day after forced weaning at Lactation Day 10, the apoptotic index was 56% lower in mammary tissue of mice that were pregnant at the time of weaning than in nonpregnant mice. Conversely, the bromodeoxyuridine-labeling index was increased sevenfold in pregnant mice compared to nonpregnant controls (3.5% vs. 0.5%, respectively). Structure of mammary alveoli was largely maintained in postweaning pregnant mice. The effect of pregnancy on three specific mammary epithelial cell survival pathways was also examined. First, pregnancy blocked the loss of Stat5a phosphorylation during involution. Significantly, loss of Stat5a phosphorylation during involution was not correlated with loss of Stat5a nuclear localization. Second, pregnancy maintained nuclear-localized progesterone receptor during lactation. Third, pregnancy was associated with increased expression of bfl-1 during involution but had little effect on the expression of other bcl-2 family members. The data indicate that pregnancy inhibits mammary cell apoptosis after weaning while permitting proliferation of the mammary epithelium, and they support the hypothesis that Stat5a and progesterone-signaling pathways act in concert to mediate this effect.