Release of immature secretory granules rich in incompletely processed proinsulin has been proposed to explain the relative hyperproinsulinemia in type 2 diabetic and insulinoma patients because of a constant secretory drive resulting from hyperglycemia and autonomous secretion, respectively. To test this hypothesis, insulin secretion was stimulated by a combination of hyperglycemia (11 mmol/L clamp), intravenous (i.v.) tolbutamide (1 g), and i.v. glucagon (initial bolus 10 micrograms/kg body weight, maintenance infusion 2 micrograms/kg body weight per hour) for 3 h. Circulating IR-insulin and IR-C-peptide concentrations increased 89-fold and 14-fold over basal values, respectively, but IR-proinsulin concentrations increased only ninefold over basal values. Estimation of the amount of insulin secreted (based on deconvolution analysis of plasma C-peptide values) showed that approximately 76 +/- 21 U were secreted during the stimulation period. This amount is a significant proportion of pancreatic insulin content in normal humans. In molar terms, IR-proinsulin (integrated incremental response multiplied by metabolic clearance rate of proinsulin) relative to IR-C-peptide (= insulin) secretion (deconvolution analysis) was estimated to be equal or even lower than the known proportion in islets (0.22 +/- 0.05%). Thus, using a near-maximal stimulation of insulin secretion maintained long enough to cause release of amounts of insulin approaching the estimated pancreatic content, no preferential release of proinsulin was observed in normal humans. Therefore, the hyperproinsulinemia of type 2 diabetes and in insulinoma patients may be caused by additional defects in the proinsulin to insulin conversion process.