A specific dog C-peptide radioimmunoassay system was established with a single antibody method using synthetic dog C-peptide as standard. The measurable range was between 0.4 pM/ml and 25.6 pM/ml. The coefficients of variation of Within and Between-assay were 6.0 and 10.8%, respectively. Dog C-peptide immunoreactivity (CPR) and immunoreactive insulin (IRI) level in the superior pancreaticoduodenal (pancreatic) vein were measured in fasting and after loading of glucose or arginine into the superior pancreaticoduodenal (pancreatic) artery. Fasting dog CPR level was 2.73 ± 0.46 pM/ml in the pancreatic vein and was 0.67 ± 0.03 pM/ml in peripheral vein. The molar ratio of CPR/IRI was 1.42 in the pancreatic vein and 6.09 in peripheral vein. Glucose (10 mg/kg/min) infusion into the pancreatic artery evoked a prompt and parallel maximal increase in CPR and IRI within 3 min. The CPR concentration increased from 3.1 pM/ml to 6.5 pM/ml and the IRI concentration increased from 1.7 pM/ml to 4.6 pM/ml at the peak. Arginine (250 mg/kg/min) infusion resulted in a prompt, parallel increase in CPR and IRI. The CPR concentration increased from 3.2 pM/ml to 5.8 pM/ml, and the IRI concentration from 1.7 pM/ml to 4.4 pM/ml at 40 sec, respectively. The insulin - to - C-peptide ratio showed approximately equimolarity.