Abnormal distribution of enteric nerves such as adrenergic, cholinergic and non-adrenergic non-cholinergic nerves (NANC) may cause the failure of relaxation at the involved bowel segment in Hirschsprung's disease (HD). Nitric oxide (NO) is a major inhibitory NANC neurotransmitter in the gastrointestinal tract. NO is synthesized by activation of nNOS (neuronal nitric oxide synthase) in the intramural ganglion cells and regulates bowel movement. To assess the distribution of nNOS in HD, immunohistochemical staining to nNOS was utilized on paraffin embedded specimens. Ten control colon specimens were tested for feasibility of staining. Immunohistochemisrty was done on ganglionic colon as well as aganglionic segment of 15 patients with HD. nNOS immunoreactivity was observed in the neuronal cells, small cells and nerve fibers in the muscle layer and submucosal neuronal cells of control specimens. This finding was also observed in the ganglionic segments of HD. But, there was no nNOS immunoreactivity in aganglionic segments of HD. In conclusion nNOS immunohistochemical staining of paraffin embedded specimen is feasible and reliable. And the results suggest that the relaxation failure of the aganglionic bowel in HD is related to the absence of nNOS containing cells and nerve fibers.