The prehepatic production of insulin in normal man was evaluated by kinetic analysis of connecting peptide (C-peptide) behavior in the plasma in men and women. Studies were performed during suppression of endogenous insulin secretion (induced by both fasting and exogenous insulin injection) as well as during stimulation of secretion (induced by oral glucose ingestion) and iv glucose injection. Least squares spline fitting of the C-peptide data by interactive computer analysis permitted evaluation of the precursor production of insulin using a two-compartment model for C-peptide removal. Basal prehepatic insulin production averaged 15-4 mU/70 kg.min in 20 subjects and was reduced to 0.9 +/- 2.2 mU/70 kg.min after 84 h of fasting. The injection of exogenous iv insulin resulted in suppression of endogenous production to 0 +/- 2.5 mU/70 kg.min. Maximum prehepatic insulin production induced by a 100-g oral glucose tolerance test was 91 +/- 1.2 mU/70 kg.min, with a cumulative hormone secretion of 11.4 +/- 2.0 U over the 5 h of observation. After the acute iv injection of 25 g glucose, production rose to 465 +/- 108 mU/70 kg.min at 2 min post injection and rapidly returned toward basal. Levels of insulin in the portal vein calculated from this analysis were markedly elevated relative to simultaneous peripheral venous levels. These results quantitate prehepatic insulin production and portal venous insulin concentration from an analysis of the behavior of C-peptide within the plasma in both the steady state and the nonsteady state in man.