Although there are more sensitive and earlier diagnostic markers for the diagnosis of acute myocardial infarction (AMI), measurement of creatine kinase (CK) MB isoenzyme (CKMB) using the immunoinhibition method is still widely used in stat laboratories. In this study, 3,290 patients with the prediagnosis of AMI underwent physical examinations, electrocardiography, and repetitive measurements of CK, CKMB activity, and CKMB mass, and 304 of them were diagnosed as having AMI. Electrophoresis of CK and CKMB mass was performed for the samples from 415 patients whose CKMB activity values were found to be increased and were not correlated with total CK levels. According to CKMB activity, CK electrophoresis, and CKMB index (100 x CKMB activity/CK) values, macro-CK (MCK) and/or increased CKBB levels were detected in 27 cases (MCK-I in 10 cases, MCK-II in 9, increased CKBB in 5, and both MCK-II and increased CKBB in 3). CKMB activity was found to be increased for all except one patient (96.3%), and the CKMB index was >25% in 25 (92.5%) of 27 cases. CKMB mass values were within the normal range in 25 of the cases with MCK. Two patients with MCK-I were diagnosed as having AMI because of increased CKMB mass and positive electrocardiography findings. The incidence of MCK and/or high CKBB levels (0.82%) in the whole group was similar to that reported for a normal population. MCK existence and increased CKBB levels may cause misleading diagnoses if CKMB mass measurements and/or CKMB index values are not used together for patients with suspected AMI.