T he in vivo hepatic metabolism of connecting peptide (C-peptide) in relation to that of insulin has not been adequately characterized. A radioimmunoassay for dog C-peptide was therefore developed and its metabolism studied in conscious mongrel dogs, with sampling catheters chronically implanted in their portal and hepatic veins and femoral artery. The hepatic extraction of endogenous C-peptide under basal conditions was negligible (4.3 +/- 4.5%) and was similar to the hepatic extraction of C-peptide measured during the constant exogenous infusion of C-peptide isolated from dog pancreas. Simultaneously measured hepatic extraction of endogenous and exogenously infused insulin were 43.8 +/- 7.6 and 47.5 +/- 4.4%, respectively. The metabolic clearance rate of infused C-peptide was 11.5 +/- 0.8 ml/kg per min and was constant over the concentration range usually encountered under physiological conditions. In additional experiments, the effect of parenteral glucose administration on the hepatic extraction of C-peptide and insulin was investigated. The hepatic extraction of C-peptide (6.2 +/- 4.0%) was again negligible in comparison with that of insulin (46.7 +/- 3.4%). Parenteral glucose administration did not affect the hepatic extraction of either peptide irrespective of whether it was infused peripherally, intraportally, or together with an intraportal infusion of gastrointestinal inhibitory polypeptide. The fasting C-peptide insulin molar ratio in both the portal vein (1.2 +/- 0.1) and femoral artery (2.1 +/- 0.3) was also unaffected by the glucose stimulus. These results therefore indicate that, since the hepatic extraction of C-peptide is negligible and its clearance kinetics linear, the peripheral C-peptide concentration should accurately reflect the rate of insulin secretion. New approaches to the quantitation of hepatic extraction and secretion of insulin by noninvasive techniques are now feasible.