The mechanism by which individual peptide and steroid hormones and cell-substratum interactions regulate milk protein gene expression has been studied in the COMMA-D mammary epithelial cell line. In the presence of insulin, hydrocortisone, and prolactin, growth of COMMA-D cells on floating collagen gels in comparison with that on a plastic substratum resulted in a 2.5- to 3-fold increase in the relative rate of beta-casein gene transcription but a 37-fold increase in beta-casein mRNA accumulation. In contrast, whey acidic protein gene transcription was constitutive in COMMA-D cells grown on either substratum, but its mRNA was unstable and little intact mature mRNA was detected. Culturing COMMA-D cells on collagen also promoted increased expression of other genes expressed in differentiated mammary epithelial cells, including those encoding alpha- and gamma-casein, transferrin, malic enzyme, and phosphoenolpyruvate carboxykinase but decreased the expression of actin and histone genes. Using COMMA-D cells, we defined further the role of individual hormones in influencing beta-casein gene transcription. With insulin alone, a basal level of beta-casein gene transcription was detected in COMMA-D cells grown on floating collagen gels. Addition of prolactin but not hydrocortisone resulted in a 2.5- to 3.0-fold increase in beta-casein gene transcription, but both hormones were required to elicit the maximal 73-fold induction in mRNA accumulation. This posttranscriptional effect of hormones on casein mRNA accumulation preceded any detectable changes in the relative rate of transcription. Thus, regulation by both hormones and cell substratum of casein gene expression is exerted primarily at the post transcriptional level.