The role of ongoing protein synthesis in mediating the posttranscriptional effects of hormones on casein gene expression in the COMMA D mouse mammary epithelial cell line was investigated using the protein synthesis inhibitors, cycloheximide and anisomycin. When COMMA D cells were pretreated with insulin and PRL for 24 h, the addition of glucocorticoids induced a greater than 20-fold increase in beta-casein mRNA accumulation with an apparent lag of greater than 8 h. Addition of cycloheximide and anisomycin not only prevented this increase, but unexpectedly, resulted in the rapid disappearance of preexisting beta-casein mRNA with a half-life of approximately 2 h. Under the same conditions, the levels of beta-actin and histone H4 mRNAs were increased markedly. In contrast, when cells were pretreated with all three lactogenic hormones for 48 h before the addition of either protein synthesis inhibitors or actinomycin D, the effects of these inhibitors on the levels of beta-casein mRNA were greatly diminished. This differential sensitivity of beta-casein mRNA to protein synthesis inhibitors was observed only in cells pretreated for greater than 24 h with all three hormones. Experiments performed in the absence of inhibitors indicated that beta-casein mRNA has a long half-life even after hormone withdrawal. These results suggest that hormone-dependent stabilization of cytoplasmic beta-casein mRNA requires ongoing protein synthesis. Cells cultured in the presence of all three lactogenic hormones slowly accumulate a labile protein(s), which exerts a selective effect on casein mRNA stability.