To investigate the diagnostic accuracy and applicability of barium enema (BE) and rectal suction biopsy with acetyl cholinesterase (AChE) histochemistry in the diagnosis of neonatal Hirschsprung's disease (HD), we retrospectively reviewed the findings of BE and AChE staining in 96 neonates with suspected HD during a 10-year period from January 1991 to December 2000. Sixty-nine cases of HD (58 males and 11 females) and 27 cases of non-HD are included in this study. In regard to BE, HD was based on definite transitional zone, suspicious HD on reversed rectosigmoid index (RSI <1), and non-HD on normal RSI (RSI>1). The histochemical criterion used for the diagnosis of HD was that of Chow et al (1977), i.e., the presence of many coarse discrete cholinergic nerve fibers in the muscularis mucosae and in the immediately subjacent submucosa regardless of infiltration of cholinergic nerve fibers in the lamina propria. Of 66 neonates with HD who underwent BE, transitional zone was identified in 33 cases (50%) and reversed RSI in 19 cases (21%), microcolon in 4 cases and normal finding in 10 cases (15%) while of 27 neonates with non-HD, there was normal finding in 16 cases and reversed RSI in 9 cases (41%). Thus diagnostic accuracy based on transitional zone was 64%. The positive predictive value of reversed RSI for the diagnosis of HD was 68%. Of 42 neonates with HD who underwent AChE histochemistry, there were 41 AChE-positive reactions and one AChE-negative reaction in a neonate with total colonic aganglionosis, while of 27 cases of non-HD, there were one equivocal AChE-positive reaction and 26 AChE-negative reactions. Thus AChE histochemical study showed a 97% diagnostic accuracy with a 98% sensitivity and a 96% specificity. In conclusion, we believe that BE is valuable as a first diagnostic step since about 80% of neonates with HD show significant radiologic findings such as a transitional zone or reversed RSI. AChE histochemical study was a more reliable diagnostic tool showing a 97% diagnostic accuracy, and is particularly valuable in neonates with HD who showed reversed RSI or normal RSI on BE. A diagnostic algorithm in neonates with suspicious HD was proposed.particularly valuable in neonates with HD who showed reversed RSI or normal RSI on BE. A diagnostic algorithm in neonates with suspicious HD was proposed.