Bovine mammary tissue from midpregnant heifers was placed in explant culture to which chlorea toxin (a potent adenylate cyclase activator) was added (0 to 100 ng/ml). Chlorea toxin increased incorporation of thymidine into DNA in the cultures; response was maximum with 10 ng/ml chlorea toxin and approximately 24 h after addition of cholera toxin. In other studies, bovine mammary tissue was transplanted subcutaneously to ovariectomized athymic nude mice. Subsequent treatment of the mice with cholera toxin alone did not affect growth of bovine mammary tissue. However, treatment with estradiol plus progesterone increased DNA synthesis in epithelial cells. In estradiol plus progesterone-treated mice, cholera toxin injections further increased DNA synthesis. In addition, estradiol plus progesterone treatment in vivo (in athymic nude mice) increased DNA synthesis of bovine mammary tissue in response to cholera toxin in vitro. This synergism between cholera toxin and ovarian steroids may have been mediated, at least in part, by estradiol plus progesterone induction of cyclic AMP dependent protein kinase, as the activity of this enzyme was increased by estradiol plus progesterone.