The localization of nitric oxide synthase, the enzyme responsible for producing the short-acting messenger nitric oxide, has been determined in the digestive tract of the rat 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 digestive tract from the esophagus to the rectum. Positive neuronal somata were very common in the myenteric ganglia. Dense positive fibers were distributed in internodal strands, the secondary plexus, the tertiary plexus, and were particularly abundant in the deep muscular plexus, while very few were observed in the submucosal ganglia. The density of these positive structures was higher in the small and large intestine than in the esophagus and stomach. The pattern of distribution suggested that some of these positive cells innervate gut muscles. Double-staining revealed that in these enteric neurons, nitric oxide synthase does not co-localize with acetylcholinesterase. Instead, vasoactive intestinal polypeptide almost always coexists with nitric oxide synthase in the myenteric plexus. Thus, nitric oxide and vasoactive intestinal polypeptide may be co-transmitters in a population of non-adrenergic, non-cholinergic neurons in the enteric nervous system.