Using a supplemental oxidation step, the authors modified the acetylcholinesterase (AChE) histochemistry technique of Karnovsky and Roots to produce staining of cholinergic nerve fibers in 10 minutes rather than in 2 hours, as required with the conventional AChE technique. They examined seromuscular intestinal biopsy specimens taken at three levels (aganglionic, transitional zone, ganglionic) from 10 patients during pull-through operations for Hirschsprung's disease, using the rapid AChE technique in addition to H&E staining. Using the acetylcholinesterase technique, the hypertrophic nerve trunks in the aganglionic segment and ganglion cells in the ganglionic segment stained strongly and were easily noticeable. The authors believe that the rapid AChE technique is a simple and reliable method for intraoperative evaluation of the extent of the aganglionic segment, because it provides quicker and more accurate identification of ganglion cells and hypertrophic nerve trunks in the space between the longitudinal and circular muscle layers of the bowel wall.