MB isoforms of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) in 848 sera obtained from 80 patients with acute myocardial infarction (AMI) were studied by agarose gel isoelectric focusing. In 173 sera (20%) from 25 patients (31%), a new isoform designated as MB3 (pI 5.4) was detected at the cathodal side of MB2 (pI 5.2) in addition to the previously known MB2 and MB1 (pI 5.1). The new isoform MB3 was found in the extract of the cardiac muscle. MB3 was dominant in the sera at an earlier stage and a shorter period of time after AMI: 1-14 h (range), 1-29.5 h and 4-154 h for MB3, MB2 and MB1 dominant, respectively. MB3 was therefore found to be an earlier and a shorter phase indicator for AMI than MB2 or MB1. However, MB2 greater than 1 was the most prevalent pattern at the time of admission to the hospital. In AMI, specificity was 96.2%, 92.4% and 90.6%, and sensitivity was 20.4%, 88.9% and 97.6%, for MB3, MB2 and MB1 isoforms, respectively. CK-MB isoform patterns were biased to MB1 dominant in the deceased group, and to MB2 dominant in the surviving group. Therefore determination of CK-MB isoforms is also useful in the course of observation of AMI. The fourth isoform, MB0 (pI 5.0), was detected at the anodal side of MB1. MB0 was a minor band of the CK-MB isoform which appeared when serum CK-MB activity increased.