Because previous reports have given inconsistent results, we re-examined the catalytic concentrations of cytoplasmic creatine kinase (CK) and of CK isoenzymes in 38 biopsies obtained from 19 different tissues. After homogenization and centrifugation many tissues showed high CK catalytic concentrations; 11 of them contained activity exceeding 50 U/g wet weight (Scandinavian recommended method). The highest specific activities were found in skeletal muscle (2400 U/g), brain (530 U/g), and myocardium (460 U/g). The separate isoenzyme activities were estimated by electrophoretic, anion-exchange chromatographic, immunoinhibiting, and radioimmunological methods. CK-BB was present in all tissues and, in fact, was the only cytoplasmic CK isoenzyme in 16 of the 19 tissues examined. CK-MM was the major isoenzyme of skeletal muscle and myocardium and was in addition observed in placenta, in trace amounts. CK-MB was present in high catalytic concentrations in myocardium (20% of total CK) and in low catalytic concentrations in skeletal muscle (1.1% of total CK).