Barium enema, anorectal manometry, and histochemical mucosal biopsy are main diagnostic procedures for chronic constipation including aganglionosis. Reliability of these examinations were evaluated quantitatively in view of the differentiation of aganglionosis. Positive caliber change in barium enema, positive rectoanal reflex in manometry, and positive nerve proliferation and/or absent of ganglion cells in biopsy were assessed as positive, and divided into two categories of true and false groups. The predictive value and efficiency were also calculated from these data. All values of barium enema were very low, suggesting that barium enema is unreliable to exact differentiation. In the neonatal period, barium enema often fails to show a caliber change (false negative), however, low predictive value of positive (a large number of false positive) was found in this series. Therefore, examination using barium enema only may lead to a misdiagnosis and overtreatment. Barium enema should be used for evaluation of extension of aganglionic area and of megarectum in cases of chronic constipation. The other two examinations, manometry and histochemical biopsy, showed excellent results for differentiation of aganglionosis: the efficiency was 98.5 in histochemical biopsy and 96.0 in anorectal manometry. Anorectal manometric study, however, is likely to be more suitable for functional evaluation of the anorectal canal than in differentiation of aganglionosis.