This study evaluates the hypothesis that cerulein relaxes the sphincter of Oddi (SO) via nitric oxide (NO). The spontaneous motility and the response to cerulein on the canine SO were recorded using a constant-perfusion technique. N(G)-L-arginine-methyl-ester (L-NAME) increased the spontaneous motility and dose-dependently reduced the cerulein-induced inhibitory response of the SO. After treatment with L-NAME at higher doses, cerulein induced an excitatory response. This effect was reversed by treatment with excess L-arginine. Similar results were obtained using cholecystokinin octapeptide in place of cerulein. In separate studies, cerulein generated increases in intracellular cAMP and cGMP levels in the SO. This indicates that the intracellular mechanism mediating cerulein-induced relaxation involves the production of cAMP and cGMP. On the other hand, treatment with L-NAME absorbed the increase in cAMP and cGMP levels by cerulein. These studies demonstrate that cerulein relaxes the canine SO mainly via NO, increasing intracellular cAMP and cGMP levels.