Nitric oxide (NO) is a potent pancreatic vasodilator, yet the pathogenic role of NO in acute pancreatitis remains controversial. NO is generated from L-arginine by NO synthase (NOS), classified into three isozymes: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). The purpose of the present study was to investigate the role of NO/NOS isozymes in the pathogenesis of cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced in male Wistar rats by two subcutaneous injections of cerulein (20 μg/kg). NG-Nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor) or aminoguanidine (a relatively selective iNOS inhibitor) was given orally, while tetrahydrobiopterin (BH4), a critical cofactor for NOS, was administered intraperitoneally 30 min before the first cerulein injection. Cerulein given repeatedly twice produced acute pancreatitis, with concomitant increases in the serum amylase level, pancreas weight, myeloperoxidase activity, lipid peroxidation and microvascular permeability. Prior administration of L-NAME, but not aminoguanidine, significantly prevented these changes, in a dose-dependent manner, and this effect was antagonized by the coadministration of L-arginine, a precursor of NO. The expression of dimetric eNOS in the pancreas was markedly suppressed by cerulein injections, together with a decrease in NO production, but the response was partially but significantly reversed by the prior administration of BH4. The increases in the serum amylase level and pancreas weight, as well as the lipid peroxidation induced by cerulein, were significantly attenuated by the administration of BH4. L-NAME had no effect on pancreatic secretion induced by cerulein. These results suggest that the uncoupled eNOS, probably caused by the decrease in endogenous BH4 availability, plays a deleterious role in the pathogenesis of cerulein-induced acute pancreatitis.