A study published in Pediatric and Developmental Pathology was based on the hypothesis that review of rectal mucosal biopsies (RMBs) taken during colonoscopy of older children with low suspect for Hirschsprung disease (HD) would frequently reveal ganglion cells (GCs). This review included 93 mucosal biopsies from children ≥1 year of age, on which clinicians had requested calretinin immunohistochemistry (CAL) on at least one specimen, from November 2013 to September 2015. The results showed that the submitted clinical indication was constipation in 75% of these patients. GCs were found within or subjacent to muscularis mucosa in 68% biopsies, 19% of which were designated from a specific anatomic site; among these, in 40%, GCs were identified on one of the first 3 sections (median 5th, range 1st-54th). On the other hand, 73% of the cases contained no or <0.5 mm of submucosa. While 98% with identified GCs showed positive CAL staining, a single case showed equivocal staining. In contrast, among the biopsies with no observed GCs, none had >1 mm of submucosa, and 70% had no submucosa. CAL was positive in 93% and equivocal/weak in 7%. The findings indicated that hematoxylin and eosin sections of RMBs can exclude HD at a specified site in many cases and provide the basis for a future study examining the utility of CAL in RMBs without submucosa as a means for excluding HD.