Mammary explants from both pregnant and virgin rats cultured in medium containing insulin, hydrocortisone and prolactin show a progressive increase in alpha-lactalbumin activity. However, when cultured with insulin and hydrocortisone only, explants from pregnant, but not those from virgin rats show an induction of alpha-lactalbumin-like activity similar to that seen when prolactin is present. The activity induced in the absence of exogenous prolactin corresponds to newly synthesized alpha-lactalbumin molecules, since 1) the activity is suppressed completely by rabbit anti-rat alpha-lactalbumin serum and 2) culture in the presence of [3H]-amino acids generates [3H]-alpha-lactalbumin, identified by SDS-polyacrylamide gel electrophoresis. Mammary tissue from virgin rats can be rendered responsive to insulin and hydrocortisone in this regard by administration in vivo of either progesterone or estrogen, but not by desoxycorticosterone acetate. This ability to convert virgin rat mammary cells to a state in which they are independent of exogenous prolactin in terms of alpha-lactalbumin synthesis represents a heretofore unreported effect of the female sex steroids.