Experimentally induced hyperinsulinemia reduces serum adrenal androgen levels in man, but does not alter cortisol secretion. To determine whether insulin might selectively inhibit adrenal androgen production by suppressing 17,20-lyase activity, ACTH-stimulated androgen secretion was assessed in 10 normal men after an insulin infusion (hyperinsulinemic-euglycemic clamp) or a control saline infusion. For the insulin clamp study, each man received a 2-U (14.4-nmol) insulin bolus dose, followed by a 2.0-mU/kg.min (14.4-pmol/kg.min) insulin infusion for 5 h. An average insulin level of 746 +/- 35 (+/- SE) pmol/L was achieved; serum glucose was maintained at 4.96 +/- 0.03 mmol/L. At the end of the insulin infusion, an ACTH stimulation test was performed, and serum steroid levels were determined 30 and 60 min later. Subjects returned 1-3 weeks later for control studies, during which 0.45% saline was infused at rates matched exactly to the rates of the dextrose and insulin infusions during the insulin clamp studies, and an ACTH stimulation test was performed after 5 h of saline infusion. After the insulin infusion, stimulation by ACTH resulted in a significant rise in the serum molar ratio of 17 alpha-hydroxyprogesterone to androstenedione (from 0.914 +/- 0.110 at zero time to 1.388 +/- 0.278 60 min after ACTH; P less than 0.05), whereas no change occurred in the ACTH-stimulated ratio of these steroids after the saline infusion (1.067 +/- 0.109 at zero time to 1.060 +/- 0.109 60 min after ACTH; P = NS). The insulin-induced change in this steroid ratio was due to a relative increase in precursor (17 alpha-hydroxyprogesterone) and decrease in product (androstenedione) responsiveness to ACTH. Similarly, insulin treatment resulted in a greater than 100% rise in the difference from baseline in the serum molar ratio of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone 30-60 min after ACTH (P less than 0.004), whereas no change in this difference was observed after the saline infusion (P = 0.71). Again, the insulin-induced change in this steroid ratio was due to a relative increase in precursor (17 alpha-hydroxypregnenolone) and decrease in product (dehydroepiandrosterone) responsiveness to ACTH. Of note, insulin treatment altered neither cortisol responsiveness to ACTH nor 17 alpha-hydroxylase activity, as indicated by similar ACTH-stimulated responses in the serum molar ratio of progesterone to 17 alpha-hydroxyprogesterone after the insulin and saline infusions (P = 0.71). Hence, the results of this study indicate that the acute elevation of serum insulin levels into the high physiological range selectively inhibits adrenal 17,20-lyase activity in man.