Analytical methods for and the clinical significance of ATP:creatine-N-phosphotransferase (EC 2.7.3.2, CK) isoenzymes have been described. The main assay methods for the isoenzymes are electrophoresis, ion-exchange chromatography and immunoinhibition. Electrophoresis is a time-consuming technique, but it is essential for the identification of CK isoenzymes. Ion-exchange chromatography is reliable for the determination of CK-MB and useful for the isolation and purification of CK isoenzymes. The disadvantages of this method are overlapping of isoenzymes with neighbouring fractions and the inevitable dilution of enzyme activity by the elution buffer. Immunoinhibition is a rapid, sensitive, specific and quantitative technique. However, mitochondrial CK and CK-immunoglobulin complex remain after the inhibition of the M subunit with the antibodies. Thus, when assessing the experimental results, it is necessary to be certain that a false positive for CK-MB has not been obtained. Since each technique has both merits and demerits, the technique most suited to the particular laboratory should be selected.