The authors studied expression of NADPH-diaphorase and NCAM in colon from 16 patients with Hirschsprung's disease (HD) and eight age-matched controls, using immunohistochemical techniques. Myenteric ganglia in the ganglionic bowel and hypertrophic nerve trunks in the aganglionic bowel displayed strong NADPH-diaphorase and NCAM reactivity. Quantitative assessment of immunoreactivity in colonic muscle demonstrated a selective absence of NADPH-diaphorase and NCAM-positive nerve fibers in the circular as well as longitudinal muscle of aganglionic bowel, whereas these immunoreactive nerve fibers were present in abundance in the muscle of ganglionic colon of HD patients as well as in the colon of controls. The lack of expression of NCAM on nerve fibers within the muscle of HD patients suggests a developmental abnormality of innervation of the muscle, and the selective absence of NADPH-diaphorase-positive nerves in the muscle is most likely responsible for the spasticity of the aganglionic segment.