CK-MB activity, which is measured by the immunoinhibition method, is an important marker for use in the diagnosis of acute coronary syndromes. In the present study, we evaluated the basal performance of a recently improved CK-MB activity kit, "L-system CK-MB," in which the activity of mitochondrial CK subunits is inhibited. Within-run and between-day precision were in the range of 1.9-2.3% and 3.2-6.0%, respectively. Diluted linearity and limit of detection were determined to be 600 U/L and 0.8 U/L, respectively. The use of L-system CK-MB allowed the inhibition of the activity of 98.1% of sarcomeric mitochondrial CK, 97.7% of ubiquitous mitochondrial CK, and 99.9% of CK-MM. The correlation coefficient (r) between CK-MB activity and CK-MB protein was 0.968. However, we found 4 cases showing CK-MB activity significantly higher than the protein concentration. Increased CK-BB activity was detected by electrophoresis in these cases. In some patients with malignant tumors, the presence of CK-immunoglobulin complex also lead to elevated CK-MB concentrations. Thus, the discrepancy in the CK-MB activity and the protein concentration may be caused by the presence of CK-BB and/or CK-immunoglobulin complex. More attention needs to be focused on samples with high CK-MB protein concentrations, especially when the CK-MB/CK ratio is high.