The murine casein locus consists of five genes, which are coordinately regulated during mammary development. The levels of casein-specific mRNAs in mammary epithelial cells increase during the second half of pregnancy and remain high during lactation. The murine γ-casein gene, which corresponds to the αS2-casein gene in ruminants, was isolated from a mouse bacterial artificial chromosome (BAC) library (strain 129SV). The gene contains 14 exons, which are distributed over 14 kb of DNA sequence. The expression pattern of the murine γ-casein gene mimics that of the neighbouring β-casein gene in terms of developmental induction in vivo. In cell culture, both the β- and γ-casein promoter are synergistically induced by prolactin and glucocorticoids. Glucocorticoid induction is critically dependent on prolactin-mediated activation of STAT5 in both promoters. Several consensus STAT5 binding sites were identified in the γ-casein promoter, some of which may have an additive effect on prolactin induction. mRNA levels of γ- and β-casein are similar in lactating mammary tissue. However, promoter segments derived from the γ-casein gene are significantly less active in cell culture than comparable fragments of the β-casein promoter. Promoter hybrids between the γ- and β-casein promoters revealed that the critical sequences which are responsible for the different in vitro activity are located in a short promoter proximal region.