Immunoreactive dog C-peptide [CPR] and insulin [IRI] concentration in the superior pancreaticoduodenal (pancreatic) vein of normal dogs were measured after administration of neurotensin, xenopsin, γ-hydroxybutyric acid [GHB], 2-Br-α-ergocryptine [CB154], pimozide, and substance-P into the superior pancreaticoduodenal (pancreatic) artery. The effect of neurotensin on B cell secretion was studied in hypophysectomized (10th to 14th day post-hypophysectomy) dogs. In normal dogs, the administration of synthetic neurotensin (10 μg/kg) induced a mild hyperglycemic response with parallel and biphasic increases in both CPR and IRI levels. In hypophysectomized dogs, basal levels of pancreatic CPR and IRI were reduced to about 14% and 55% of the control, respectively. Neurotensin had little effect on pancreatic B cell secretion. Administration of synthetic xenopsin (10 μg/kg) caused mild hyperglycemia and rapid equimolar increases of CPR and IRI in the pancreatic vein. GHB had suppressive effects on both CPR and IRI secretion. Administration of a high dose (500 mg/kg) of GHB showed a more marked decrease in CPR and IRI than that of the low dose (100 mg/kg). The degree of suppression in CPR and IRI secretion was nearly equimolar. A bolus injection of CB154 (200 mg/kg) had caused mild hyperglycemia and biphasic CPR and IRI increases, but after premedication with pimozide (1 mg/kg, i.m.) the same dose of CB154 administration resulted in decrease of CPR and IRI concentrations in the pancreatic vein. With infusion of synthetic substance-P (50 ng/kg/min) for 30 min, CPR and IRI concentrations in the pancreatic vein were reduced parallel and were nearly equimolar. The above findings indicated that C-peptide and insulin were released from the B cell in equimolar concentrations after stimulation by various CNS affecting agents.