Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase-containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons.