We studied the internal anal sphincter (IAS) muscle from 10 patients with achalasia and five normal controls using histochemical staining for NADPH-diaphorase and acetylcholinesterase (AChE). Normal control IAS muscle contained occasional AChE-positive nerve fibers, whereas IAS achalasia specimens demonstrated prominent AChE-positive nerve fibers in muscle layers. NADPH-diaphorase activity was strongly expressed in nerves in the normal IAS muscle but was absent or scanty in the muscle of patients with IAS achalasia. Our findings of increased AChE-positive nerves and the absence of NADPH-diaphorase activity taken in conjunction with reports of abnormal peptidergic innervation indicate that complex neural abnormalities occur in IAS achalasia. The primary event remains obscure, but it is possible that a single defect, such as nitrergic nerve depletion, may lead to compensatory changes in the other nerve fibers.