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Background and study aims Acute pancreatitis is still the most common complication after endoscopic sphincterotomy (ES) and cholangiopancreatography (ERCP). The aim of this study was to detect the time when the peak of serum amylase was predictive for postprocedure pancreatitis or long-lasting severe hyperamylasemia, in order to plan the follow-up of patients. Methods Serum amylase activity was measured in a prospective series of 409 consecutive patients after ES, immediately before ES and two, four, eight and 24 hours thereafter; the two, four and eight-hour data were compared with those at 24 hours and with the outcome. Evaluation was done separately for the 198 cases with pancreatic duct opacification and for the 202 cases at high risk for postprocedure pancreatitis. Results Twenty-four hours after ES, amylase was still more than five times the upper normal limit in 26 patients, associated with pancreatic-like pain in 19 of them (mild/moderate pancreatitis) and asymptomatic in the remaining seven (long-lasting severe hyperamylasemia). There was a significant difference at all sampling times between the 26 patients with 24-hour severe hyperamylasemia and those with the lower level. Although the sensitivity of amylase measurement in detecting pancreatitis was highest at eight hours, in practice the four-hour assessment appears a reliable predictor. Almost all patients with serum amylase levels more than five times the upper normal limit at four, eight and 24 hours had had pancreatic duct opacification. In contrast, patient-related risk factors for postprocedure pancreatitis did not play a significant role in the present series. Conclusions Serum amylase assessment four hours after ES is a reliable, cost-effective follow-up and minimizes the likelihood of underestimating the risk of post-procedure pancreatic reaction. It should be recommended particularly in out-patients and when pancreatic duct opacification has occurred.