The effect of insulin and its interaction with intracellular messenger systems on in vitro inhibin production by adult rat isolated seminiferous tubules has been investigated using a recently developed inhibin radioimmunoassay (RIA). Seminiferous tubule segments (5 cm) from intact adult rats were exposed to insulin (0.05-5000 ng/ml) for 2 days of culture. Insulin caused a dose-dependent inhibition of basal inhibin secretion with reversal of this inhibition at very high doses (5000 ng/ml). The ability of follicle-stimulating hormone (FSH) to induce inhibin secretion was also inhibited by insulin (50 ng/ml). Insulin reduced the stimulation of inhibin production by dibutyryl cyclic AMP (dbcAMP) and this effect was prevented by the addition of theophylline (0.4 mM), while theophylline alone was unable to prevent the effect of insulin on basal inhibin secretion. Phorbol 12-myristate 13-acetate (PMA) mimicked the effect of insulin reducing basal and FSH-induced secretion of inhibin. No additive effects on basal inhibin secretion were observed with a combination of PMA and insulin. Ethylenediaminetetraacetic acid (EDTA, 2 mM) significantly reduced basal and FSH-induced inhibin production, while the combined effects of EDTA and insulin on basal and FSH-induced inhibin production were additive. These data demonstrate an inhibitory effect of insulin on inhibin production by isolated seminiferous tubules mediated via at least two mechanisms namely the inhibition of the cAMP-protein kinase A system and stimulation of protein kinase C activity. © 1989.