Monolayer cultures of mammary gland epithelial cells were prepared from the abdominal glands of midpregnancy mice. After collagenase digestion of mammary tissue and separation by differential centrifugation, the isolated epithelial cells were cultured in Eagle's Minimal Essential Medium supplemented with 10% fetal bovine serum and insulin (6 micrograms/ml). Six days later, when the cultures were in log growth and nearly confluent, the effects of insulin and/or hydrocortisone on the rates of RNA, DNA, and protein synthesis were determined in a serum-free medium. At physiological concentrations, insulin enhanced the rates of uptake and incorporation of [3H]uridine into RNA, of [3H]thymidine into DNA, and of [3H]leucine into protein. Hydrocortisone was shown to be biphasic with regard to concentration in attenuating or augmenting insulin's effects on macromolecular synthesis.