The isoforms of CK-MB have recently received attention as potential biochemical markers for the early diagnosis of an acute myocardial infarction. A sensitive (analytical sensitivity = 0.2 ng/ml) immunochemical mass assay for the direct measurement of the CK-MB2 isoform has been developed by us. This assay utilizes a specific monoclonal capture antibody directed against the B-subunit of CK-MB and a specific monoclonal antibody conjugate directed against the CK-M + lysine subunit. Owing to the lack of a World Health Organization standard for CK-MB, the percent CK-MB2 values had to be normalized by determining both CK-MB and CK-MB2 in assays which differ only in the specificity of the anti-CK-M conjugate. Thus, a related CK-MB immunochemical mass assay utilizing the identical capture antibody and a specific monoclonal antibody conjugate directed against the CK-M subunit was also developed. Analytical sensitivities of the CK-MB and CK-MB2 assays were determined to be 0.5 ng/ml and 0.2 ng/ml, respectively. Both CK-MB and CK-MB2 levels were determined in 46 hospitalized non-AMI patients and 35 non-hospitalized normal patients. Of the 46 hospitalized non-AMI patients (mean age = 70), 26 percent had either CK-MB and/or CK-MB2 values below the level of sensitivity of the CK-MB or CK-MB2 isoform assays. For patients with CK-MB values between 0.5 ng/ml and 2.99 ng/ml, the percent CK-MB2 values ranged from 35 percent to 97 percent. For patients with CK-MB values between 3.0 ng/ml and 6.5 ng/ml, the percent CK-MB2 values ranged from 23 percent to 72 percent. Similar results were obtained for the non-hospitalized group. This assay appears to be useful in determining the clinical utility of CK-MB2.