The regulation of PRL production in GH3 rat pituitary tumor cells and normal rat pituitary cells exposed to insulin was studied. Cells were treated with semisynthetic human insulin for up to 5 days. Insulin (0.7 nM) stimulated PRL production by 30%, and cortisol (100 nM) suppressed it by 80% in GH3 cells. Insulin (3.5 nM) partially blocked the suppression of PRL secretion induced by up to 100 nM cortisol. Equimolar doses of insulin-like growth factor I failed to consistently stimulate either basal PRL secretion into the medium or cortisol-suppressed PRL in GH3 cells. A 65 nM concentration of insulin-like growth factor I was required to stimulate basal PRL secretion in GH3 cells. Relative levels of PRL mRNA sequences in both GH3 cells and normal rat pituitary cells were stimulated by insulin. When cells were incubated with cortisol (10 nM), PRL mRNA levels were suppressed to 40% of control values. Simultaneous incubation of cells with insulin (3.5 nM) partially reversed the cortisol-induced suppression of PRL mRNA to 60% of control values. The results show that insulin antagonizes cortisol-induced suppression of PRL. Physiological doses of insulin stimulate PRL production and PRL mRNA levels, suggesting a direct effect of insulin on either PRL gene transcription or stabilization of PRL mRNA in these cells.