These studies were designed to further elucidate the relative contributions of transcriptional and posttranscriptional mechanisms involved in beta-casein gene regulation in the mammary epithelial cell line designated COMMA-D and a clonal subline desginated HC-11. Primary transcripts were mapped under various hormonal and substratum conditions using the technique of nuclear run-on transcription and single stranded sense and antisense probes spanning the beta-casein gene. In the presence of insulin alone very little sense transcription is detectable, but antisense transcription is observed, which originates at least 150 basepairs upstream of the normal start site of transcription and is present regardless of hormonal, cell substratum, cell type, or gene activity. Antisense transcription is also detectable in the 3' end of the gene. Insulin, glucocorticoids, and PRL are all necessary for a maximal increase in transcription. A 2- to 4-fold increase in transcriptional activity is observed in the presence of insulin and PRL compared to insulin alone, and this is accompanied by a 125-fold increase in the level of beta-casein mRNA. All three hormones act synergistically to induce a 10-fold increase in transcriptional activity, but the transcriptional increase across the gene is not equimolar. The 5' half of the gene is transcribed at a level that is 2- to 10-fold lower than that of the 3' half of the gene. These studies reveal a significant transcriptional component to beta-casein gene regulation which was not heretofore detected using double stranded cDNA probes representative of only the 3' half of the gene.