In order to study the hormonal regulation of gene expression in mammary epithelial cells, we isolated a prolactin-responsive cell clone, HC11, from the COMMA-1D mouse mammary epithelial cell line. Clone HC11 was selected as a unique example of a cloned mouse mammary epithelial cell which has no requirement for complex, exogenously added, extracellular matrix or co-cultivation with other cell types for the prolactin-dependent in vitro induction of the endogenous beta-casein gene by lactogenic hormones. Induction of beta-casein mRNA is rapid and was detected 3 h after hormone stimulation. A prolactin-dependent increase in the rate of transcription of the beta-casein gene was shown in an in vitro nuclear transcription assay. beta-Casein protein was detected in an immunoblot assay after 24 h, and further accumulated during 5 days of hormone treatment. To identify low-abundance proteins induced directly after prolactin stimulation, mRNA was accumulated during 5 h of stimulation of HC11 cells with prolactin in the presence of cycloheximide. Following cycloheximide removal, the mRNA was translated into protein during a 60-min [35S]methionine pulse and the proteins were resolved by DEAE ion exchange HPLC and SDS-PAGE. A strong induction of a 120-kd cytosolic protein was detected which was maximally expressed within 6 h of hormone stimulation.