To determine the accuracy of the serum amylase in identifying a pancreatic source, amylase isoenzymes were determined prospectively in 65 patients initially evaluated with a complaint of abdominal pain and associated hyperamylasemia. Isoenzyme patterns were demonstrated by an electrophoretic technique, and the results were correlated with clinical diagnoses. Patients were divided into two diagnostic groups. Group I consisted of 42 patients with clinical findings suggesting pancreatitis. P-type isoenzymes were normal or elevated in 31 of these patients (74%), and s-type isoenzymes were normal or elevated in 11 (26%). Group 2 consisted of 23 patients with abdominal pain attributed to causes other than pancreatitis. Four patients (17%) had elevation of p-type isoenzymes, and 19 patients (83%) had predominantly s-type patterns. We conclude that amylase isoenzymes cannot determine unequivocally the cause of hyperamylasemia, but they can enhance the diagnostic specificity of the serum amylase. Elevated serum amylase with a predominant p-type pattern suggests pancreatic disease; elevation of s-type isoenzymes suggests but is not conclusive for, diagnoses other than pancreatitis. Hyperamylasemia with a normal isoenzyme pattern occurred in a few patients in both groups, and it was nondiagnostic.