As a possible step to evaluate the role of tumor promoters in normal cells, the effects of phorbol and its ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on normal mammary gland function and pituitary secretion of growth hormone (GH) and prolactin, were examined in lactating C3H/He mice. Daily subcutaneous injections of 400 μg phorbol or 4 μg TPA during Days 12 and 14 of lactation resulted in a significant decline in litter growth rate and the ratio of RNA synthesis: DNA synthesis of mammary glands associated with an increase in mammary gland DNA synthesis. On the other hand, pituitary synthesis and release of GH and prolactin, the pattern of estrous cycles and ovarian structures were affected little by these treatments. All findings strongly suggest that these tumor promoters can directly inhibit lactation by shifting mammary gland cells from a functional state to a mitotic state.