The cellular localization of nitric oxide synthase, the enzyme responsible for producing the inorganic gaseous messenger nitric oxide, has been studied in the biliary ducts of the mouse using histochemistry for reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity, a specific marker for neuronal nitric oxide synthase. Positively stained neurons were found throughout the entire biliary ducts from the hepatic ducts to the opening of the duodenal papilla. Positive neuronal somata were located in ganglionic plexuses of both the gallbladder and the pancreas. In addition, positive neuronal somata were also found as disseminated ganglionic cells throughout the entire biliary ducts including the duodenal papillary sphincter. Positive nerve fibers were found not only in regions around these ganglionic cells but also in the walls of blood vessels throughout the entire biliary ducts. The pattern of distribution suggested that some of these positive fibers innervate biliary muscles while some other fibers innervate vascular walls. Double-staining revealed that in the gallbladder plexus nitric oxide synthase does not co-localize with acetylcholinesterase, white that in the pancreatic plexus nitric oxide synthase almost always co-localizes with acetylcholinesterase. It is therefore suggested that nitric oxide may be a transmitter acting in non-adrenergic, non-cholinergic neurons in upper portions of biliary ducts such as the gallbladder.