Creatine kinase isoenzymes in cytosolic and mitochondrial fractions from human cardiac tissues were studied by analytical and preparative isoelectric focusing (IEF), electrophoresis and immunoinhibition. Analytical IEF on agarose gels revealed many creatine kinase variants in human cardiac cytosol prepared by extraction with a hypotonic medium. The bands located at approximately pH 5.5 were shown to contain creatine kinase-MB and minute creatine kinase-BB bands by electrophoresis. Two bands which focused closely together in IEF (pH 6.85-7.0) showed an electrophoretic migration pattern similar to creatine kinase-MM. One of them (IP 6.85) showed a complete inhibition by anti-creatine kinase-M antibodies, whereas the other showed only 50% inhibition. Increasing the salt concentration of tris-HCl (0.1 mol/l) in the extraction medium resulted in additional creatine kinase variants. They were characterized by high alkaline isoelectric points and were not inhibited by anti-creatine kinase-M antibodies. These variants corresponded to two cathodic bands in electrophoresis. The treatment of washed mitochondria with phosphate buffer resulted in a release of mitochondrial variants with different isoelectric points, as shown by analytical IEF in agarose gels. The same pattern was obtained by using preparative IEF. Variants with high alkaline isoelectric points gave rise to two cathodic bands upon electrophoresis. These two bands resembled those present in cytosol after extraction with high salt concentration. No complete inhibition with anti-creatine kinase-M was observed in any of the eluates. The mitochondrial variants exhibited different affinities towards creatine phosphate and ADP. Variants with higher alkaline isoelectric points showed lower Km-values for these substrates than those with less alkaline isoelectric points.