We compared the clinical sensitivity, specificity, and diagnostic efficiency of measuring creatine kinase-3 (MM) isoenzyme sub-types (CK, EC 2.7.3.2) with the measurement of CK-2 (MB) isoenzymes for the diagnosis of acute myocardial infarction. Serial blood collections at 3-h intervals from 35 patients with acute myocardial infarction were examined. In attempts to reperfuse their coronary arteries, some of these patients were treated with pharmacological thrombolysis (streptokinase, tissue plasminogen activator), with or without coronary angioplasty. The infarction patients were divided into two groups: patients who were successfully treated with thrombolytic agents (i.e., they achieved coronary reperfusion), and patients who were treated unsuccessfully or who were not treated acutely. We also examined blood from 34 non-infarction patients. We measured CK-3 sub-types by both anion-exchange liquid chromatography and a modified high-voltage electrophoresis method, and CK-2 by immunoprecipitation. Our results show that during the first few critical 3 to 9 h after onset of chest pain, measurement of CK-3 sub-types has the highest diagnostic efficiency; in contrast, CK-2 has the highest efficiency during the 10- to 21-h time intervals. Thus early diagnosis of acute myocardial infarction can be based on rapid assays of CK-3 sub-types.