Hirschsprung's disease (HD) is a congenital malformation of the neural innervation of the large intestine that affects normal peristaltic action and causes obstructive constipation in neonates. A narrowed segment of distal large bowel of variable length lacks neuronal ganglion cells, and the normally innervated bowel upstream becomes dilated. The gold standard for the pathologic diagnosis of HD has traditionally consisted of microscopic evaluation of rectal tissue for the absence (lack) of ganglion cells and an increased acetylcholinesterase staining pattern. The following article describes a protocol that optimizes the handling and processing conditions of suction rectal mucosal biopsies so that an accurate diagnosis can be made, emphasizing a detailed gross and microscopic examination. Once the diagnosis is established, mapping identifies a normally innervated bowel segment that can be used for repair. Even among Children's Hospitals, there is variation in the diagnostic approaches taken in rec...