Abstract The origin of an atypical creatine kinase (CK, ATP:creatine W-phosphotransferase, EC 22.214.171.124) migrating cathodic to the MM position found in the serum of a cancer patient was studied. The electrophoretic mobility of the atypical CK is similar to that of the fast-moving cathodal mitochondrial CK. The relative molecular mass was estimated to be approximately 350000, and was similar to that of the fast-moving cathodal mitochondrial CK. The atypical CK reacted with anti-human mitochondrial CK antibody. It is therefore suggested that the atypical CK is of mitochondrial origin. After incubation in 2 mol/l urea, the enzyme was converted into a new form migrating to the MM position. The conversion was observed in liver mitochondrial CK but not heart mitochondrial CK. The residual CK activity after heating at 56°C for 60 s was 77%, and the apparent K m value for creatine phosphate at 30°C was about 0.27 mmol for the atypical CK. These characteristics were very similar to those of the liver mitochondrial CK, because the data from the enzyme determined at the same time were 75% for residual enzyme activity to heat, and 0.24 mmol for apparent K m value. Therefore liver mitochondria are suggested to be the source of the atypical CK.