Following myocardial damage as in acute myocardial infarction (AMI) or open heart surgery, the tissue damage might result in a release of mitochondrial CK (CK-MIT). The presence of this CK isoenzyme in serum may be detected after chromatographic separation of CK-activity on Sephacryl S-200. By combining chromatographic separation of CK-MB with immunologic inhibition of CK-M, both CK-MB and CK-MIT can be estimated in serum. Using this procedure changes in enzyme activities were studied in ten patients with AMI and twelve patients subjected to open heart surgery using cardioplegia. Following AMI CK-MB peaked about 24 h after onset of ischaemic symptoms. CK-MIT increased similarly and reached a plateau after 24 h where it remained during an additional 24-36 h. At peak CK-MB concentration, the corresponding CK-MIT activity was about 22% of the CK-MB activity. Following cardiac surgery there was a rapid release of CK-MB with a peak about 5 h after release of aortic cross-clamping, and with a simultaneous CK-MIT activity amounting to 19% of the CK-MB activity. In conclusion, CK-MIT is released into serum following myocardial ischaemia. Its appearance has time characteristics similar to that of other mitochondrial enzymes. The CK-B method does not specifically determine CK-B, but non-CK-M, which in cardiac ischaemia at peak serum CK-MB concentrations includes about 20% CK-MIT.