Physiological doses of insulin were shown by us to suppress basal and hydrocortisone (HCT)-induced GH secretion and GH mRNA levels in GH3 rat pituitary cells. Because T3 stimulates GH gene expression, the effects of semisynthetic human insulin were tested on T3-induced GH secretion. Cells were first incubated for 48 h in medium containing insulin and fetal calf serum (10%) depleted of T3 and T4 by ion exchange resin. Insulin suppression of basal GH secretion was independent of T3, as insulin (0.7 nM) suppressed basal secretion of GH by 40% in T3-depleted cells. Medium glucose concentrations and cell proliferation did not differ in control or insulin-treated wells. The 4-fold increase in GH secretion induced by added T3 (0.5 nM) during 72 h was suppressed up to 40% by insulin (P less than 0.001). This suppression was maximal with 3.5 nM insulin and occurred after a lag period of 48 h. The previously described 20-fold synergistic stimulation of GH by T3 (0.5 nM) together with HCT (1 microM) was also suppressed by insulin (3.5 nM) by 80% during 72 h of incubation. The selectivity of the inhibitory effects of insulin on GH were also shown when the T3-induced suppression of PRL secretion was reversed by insulin treatment. The data show that physiological doses of insulin antagonize T3-induced stimulation of GH secretion and also partially block the synergistic stimulation of GH by T3 and HCT. As T3 and HCT probably stimulate GH gene transcription at different sites, insulin may suppress GH gene expression at a more distal site. Alternatively, the inhibitory effects of insulin on T3-induced GH secretion in these cells may be posttranscriptional.