Mammary epithelial cells isolated from pregnant mice were plated on a collagen gel matrix and cultured in a serum-free medium supplemented with combinations of insulin, dexamethasone and prolactin (PRL). After the cells formed a monolayer, the collagen gel was detached from the culture dish and allowed to float in the medium. Quantification of gamma-casein mRNA by a competitive PCR method revealed that the cells on the floating gel accumulated considerably larger amounts of gamma-casein mRNA than the cells on the gel remained attached to the dish. Under these floating collagen gel culture conditions, addition of both dexamethasone and PRL to the insulin-supplemented basal medium maximally stimulated the accumulation of gamma-casein mRNA. These observations strongly suggest that the status of the extracellular matrix as well as hormones controls the differentiation of mouse mammary epithelial cells at the mRNA level.