Intraoperative biopsies are essential for accurately distinguishing between Hirschsprung's disease (HD) and intestinal neuronal dysplasia (IND), and vital for determining the extent of abnormal bowel for surgical correction. IND can be associated with HD and can be a cause of postoperative complications. Routine hematoxylin and eosin (H&E) staining is sometimes inadequate for identification of ganglion cells in biopsy specimens and can be the cause of confusion. The authors found synaptophysin (SY) to be more specific as an indicator of abnormal bowel innervation and invaluable for surgical planning. Twenty patients (15 with HD, 3 with IND, and 2 with IND complicating HD) received biopsies intraoperatively. There was markedly reduced immunoreactivity (ie, a decreased number of SY-positive synapses) seen in the intestinal smooth muscle layers of transitional, aganglionic, and IND bowel segments, whereas immunoreactive synapses were abundantly present in the smooth muscle layers of ganglionic colon in HD. SY immunoreactivity also showed ganglion cells and hypertrophic nerve trunks clearly. Rapid SY staining is a simple and consistently reliable method for the intraoperative evaluation of the distribution of synapses in myenteric plexuses as well as smooth muscle layers.