Ovariectomized mice were either sham operated or sialoadenectomized and injected daily for 18 days with saline, estradiol + progesterone, cholera toxin or estradiol + progesterone+cholera toxin. Mammary development score and DNA were increased by estradiol + progesterone, but not by cholera toxin alone. In combination with estradiol + progesterone, cholera toxin increased mammary development score and mammary DNA. Sialoadenectomy reduced the ability of estradiol, progesterone and cholera toxin to induce mammary development. In other experiments, mice were primed with estradiol + progesterone for 10 days, and mammary tissue removed for in vitro culture with various combinations of insulin, aldosterone, cholera toxin and epidermal growth factor. In combination with insulin and aldosterone, cholera toxin increased mammary development in vitro. Sialoadenectomy reduced the ability of cholera toxin to induce mammary development in vitro. The effect of sialoadenectomy on mammary development was alleviated by adding epidermal growth factor to culture medium. Biochemical studies indicated that sialoadenectomy reduced the ability of estrogen and progesterone to induce cyclic AMP dependent protein kinase levels in mammary tissue, and also the ability of cholera toxin to induce accumulation of cyclic AMP in tissues. These effects of sialoadenectomy were reversed by addition of EGF to culture media.