Despite having an optimal pull-through operation, some children with Hirschsprung's disease (HSCR) continue to experience persistent bowel symptoms. Coordinated colonic electrical activity depends on intercellular communication between the enteric nerves, interstitial cells of Cajal (ICCs), smooth muscle cells, and fibroblast-like (platelet-derived growth factor receptor-alpha-positive) cells. Gap junctions are low-resistance channels composed of connexin (Cx) proteins which couple cells electrically and chemically. We aimed to evaluate the expression patterns of Cx26, Cx36, and Cx43 in the colon of children with HSCR and in healthy control colon.Entire resected colonic specimens were collected from children undergoing pull-through surgery for HSCR (n = 10). Colonic controls were collected at colostomy closure in children with imperforate anus (n = 8). Distribution of Cx26, Cx36, and Cx43 was assessed using double-labeled immunofluorescence and confocal microscopy. Protein expression was quantified using Western blot analysis.Sparse punctate Cx36 expression was seen in the myenteric plexus in nerve trunks and some platelet-derived growth factor receptor-alpha-positive cell and ICC fibers. Cx26 was similarly distributed, although it was not coexpressed in ICCs. Cx43 was only coexpressed with ICCs in the myenteric plexus. Expression of Cx26 and Cx43 was markedly reduced in the aganglionic colon in HSCR compared to controls, while Cx26 expression was also moderately reduced in the ganglionic bowel in HSCR.Reduced expression of Cx26 and Cx43 is implicated in the pathophysiology of colonic dysmotility in the aganglionic bowel as well as, in the case of Cx26, the ganglionic bowel in HSCR.