Hirschsprung's disease (HD) is defined as a congenital absence of ganglion cells in the distal bowel. Functionally, there is a loss of enteric neuromuscular inhibition. Inhibitory intestinal innervation includes extrinsic nonadrenergic, noncholinergic (NANC) nerves. Nitric oxide (NO) is proposed to be a NANC neurotransmitter. Sites of NO synthesis can be localized using a NO-dependent nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical assay. We present a study of the distribution of NO neural elements in patients with HD. Routine hematoxylin-eosin (HE) histology as well as histochemical localization of NO synthase activity was carried out on fixed laminae and sectioned tissue of infant colon. NO synthase positive nerve cells and fibers were found throughout the wall of the proximal ganglionated colon. In the myenteric plexus disposition of these nerves parallels the known NANC innervation. "Aganglionic" distal colon displayed disrupted ganglia and increased nerve fibers. Selective preservation of NO synthesizing neurons was also seen. Punctate labeling of an apparent nonneuronal origin was also noted on the surface of arterioles. NO stain simplifies the pathological diagnosis of HD. The presence of NO positive nerve cells in HD suggests that aganglionosis is a misnomer. The lack of characteristic HE findings in other forms of neuronal intestinal dysplasia indicates the need for routine simple, more sensitive neural staining of colonic biopsies in selected infants with constipation.