BACKGROUND The purpose of this study was to determine whether nitric oxide regulates insulin secretion in the isolated perfused human pancreas. METHODS Single-pass perfusion was performed in four pancreata with a modified Krebs medium. Sequential 10-minute infusions (separated by 10-minute basal periods) of (1) 25 nmol/L acetylcholine, (2) 2.5 mumol/L acetylcholine, and (3) 16.7 mmol/L glucose were initially infused. Then 0.1 mumol/L of NG-monomethyl-L-arginine (NMMA) was infused during a period of 10 minutes, and steps (1) through (3) were repeated. The change in insulin secretion from basal levels during each stimulation was calculated and compared with that seen after NMMA infusion. RESULTS Infusion of 25 nmol/L and 2.5 mumol/L acetylcholine resulted in a significant stimulation of insulin secretion before NMMA infusion (p < 0.05) and after NMMA infusion for acetylcholine at 25 nmol/L (p < 0.05). There was a significant decrease in acetylcholine-induced insulin secretion after NMMA infusion for acetylcholine at 25 nmol/L and 2.5 mumol/L compared with before NMMA infusion (p < 0.05). Infusion of 16.7 mmol/L glucose significantly stimulated insulin secretion before and after NMMA infusion, but there was no significant difference seen with insulin secretion before and after NMMA infusion. Insulin secretion was significantly inhibited during NMMA infusion (p < 0.05). CONCLUSIONS These data show that infusion of the nitric oxide synthase inhibitor NMMA suppressed cholinergic-stimulated insulin secretion but did not affect glucose-stimulated insulin secretion. We conclude that nitric oxide regulates insulin secretion in the isolated perfused human pancreas.