Using a modification of the histochemical acetylcholinesterase (ACE) reaction of Karnovsky and Roots, 154 suction rectal biopsies performed during the clinical investigation for Hirschsprung's disease were examined and compared to simultaneously obtained paraffin-embedded, hematoxylin-eosin (H&E)-stained tissue. Serial paraffin sections were adequate to identify normal ganglia or to raise suspicion of Hirschsprung disease at initial biopsy in 129 of 154 (83%) cases. Interpretation of the ACE-stained tissue correlated exactly with this interpretation in 123 instances; 6 cases had uninterpretable ACE frozen sections. From this group of adequate paraffin sections, 18 patients with aganglionic megacolon were identified with the ACE preparation, relying primarily on a pattern of unusually thick and numerous nerve fibers within the muscularis mucosa. Twenty-five paraffin-embedded specimens were deemed uninterpretable because of absent or insufficient submucosa or because of location. The ACE-stained section, however, correctly predicted the presence or absence of ganglia in 23 of these additional cases where the diagnosis could subsequently be confirmed either by repeat biopsy or repeated clinical follow-up examination of the patient. Using the ACE method alone, therefore, a diagnosis was obtainable at initial biopsy in 146 of 154 specimens (95%). This percentage was increased to 99% (152 of 154 specimens) if both ACE and paraffin sections were utilized. Recognition of the normal and abnormal patterns of nerve fiber staining in ACE preparations permits reliable, decisive interpretation of rectal suction biopsies in the evaluation of Hirschsprung's disease.