During the lactation period, mammary epithelial cells secrete large amounts of milk proteins. The coordinate regulation of milk protein expression is effected by the lactogenic hormones. We have investigated the activity of a mammary gland-specific transcription factor (MGF), which mediates hormonal influences at the level of a milk protein gene promoter. MGF-binding sites are present in the promoters of the most abundantly expressed milk protein genes. Mutation of the MGF-binding site in the beta-casein gene promoter completely abolishes responsiveness of the promoter to lactogenic hormones in cultured mammary epithelial cells. MGF activity is closely controlled in vivo. High MGF levels were found in mouse mammary gland nuclear extracts toward the end of pregnancy and during lactation. Withdrawal of suckling pups from their mothers during the lactation period caused a strong and rapid decrease of MGF activity. Readdition of pups to their mothers restored maximal MGF levels within 4 hr. We investigated MGF phosphorylation as a possible posttranslational modification responsible for regulation of the DNA-binding activity of MGF. Treatment of nuclear extracts from lactating mammary glands with potato acid phosphatase abolished MGF-binding activity. Casein kinase II phosphorylation of nuclear extracts from animals withdrawn from their pups for 24 hr enhanced MGF-binding activity. These results suggest that the reversible activation of MGF by suckling and withdrawal might be mediated by the action of kinases and phosphatases.