Nitric oxide-containing nervous structures were localized in the human colon using NADPH diaphorase activity and nitric oxide synthase immunoreactivity. We found some, solitary NADPH diaphorase-reactive and nitric oxide synthase-immunoreactive neurons in the submucous plexus, while the myenteric plexus contained several neurons, often arranged in clusters, and nerve fibers showing these markers. The circular muscle layer contained a dense plexus of NADPH diaphorase-reactive nerves, which was greater than that in the longitudinal muscle layer. We report on co-localization of NADPH diaphorase activity and VIP immunoreactivity in several neurons of the myenteric ganglia. Such co-localization has not been reported previously for human colon. Localization of nitric oxide synthase and VIP in the myenteric plexus and in the nerves of circular muscle layer raises the possibility that nitric oxide contributes to the regulation of motility in the human colon.