The protein synthesis inhibitor cycloheximide (Chx) suppresses prolactin-induced beta-casein gene expression in the mammary epithelial cell line COMMA-D. As the mechanism underlying this effect is unclear, the effects of protein synthesis inhibitors on interactions of transcription factors with the beta-casein promoter were examined. Suppression of prolactin-induced beta-casein gene expression occurred in both COMMA-D cells and primary mammary cell cultures with as little as 2 h protein synthesis inhibition. This was associated with changes in transcription factors interacting at a response element in the proximal region of the rat beta-casein promoter. Inhibition of protein synthesis was associated with NF-kappaB binding at a site immediately 3' to the Stat5-binding site at position 97-89 of the beta-casein promoter, suppression of Stat5 DNA-binding activity, and inhibition of Stat5 tyrosine phosphorylation. Treatment with the NF-kappaB inhibitor parthenolide failed to restore prolactin responsiveness. These results show that protein synthesis inhibition is associated with both blockage of prolactin-Stat5 signaling and NF-kappaB binding to the beta-casein promoter, but that the latter is not necessary for the suppression of beta-casein expression.