An improved staining technique for acetylcholinesterase (AChE) activity using rubeanic acid was used to make a clinical diagnosis in 54 children with constipation. Nineteen were thus confirmed to have Hirschsprung's disease. False positive or negative reactions were nil. The sites of AChE activities were in the form of black deposits and the contrast was sharp. This approach should find a wide application for the diagnosis of Hirschsprung's disease.