The authors studied rectal suction biopsy material in 60 consecutively treated patients suspected of having Hirschsprung's disease (HD) (age range, 3 days to 12 years). According to acetylcholinesterase (AChE) staining, 10 patients had HD, three had neuronal intestinal dysplasia (NID), and 47 were normal. The diagnoses were confirmed by H&E staining of biopsy material and by examination of surgically resected material in the HD and NID cases. The 60 cases were stained with a monoclonal antibody to the nerve growth factor receptor (NGFR) using immunohistochemistry on fresh frozen biopsy tissue. In the 47 normal biopsy specimens, there was a large number of immunoreactive fibers in the lamina propria, and staining of the muscularis mucosae and submucous ganglia. In contrast, there were no immunoreactive fibers in the lamina propria in patients with HD and NID. A striking finding was the strong expression of immunoreactivity on the perineurium of submucosal hypertrophic nerve trunks in cases of HD. The results indicate that NGFR immunoreactivity is similar in specificity and sensitivity to AChE in the diagnosis of HD. Because the technique uses an immunocytochemical rather than a histochemical technique and the results were easier to interpret, NGFR staining may be an important additional technique to diagnose HD.