Two types of mitochondrial creatine kinase (Mi-CK), sarcomeric (sMi-) and ubiquitous (uMi-)CKs, were isolated from normal human cardiac muscle and brain tissue, respectively, and their heterogeneity was characterized by means of isoelectric focusing (IEF). Octameric sMi-CK and uMi-CK were electrophoresed cathodic to cytoplasmic muscle-type creatine kinase isoenzyme (CK-MM) and dimeric Mi-CKs were found at the position of CK-MM on a cellulose acetate membrane. The electrophoretic mobilities of sMi-CK were similar to those of uMi-CK. Octameric sMi-CK was focused at pI7.1-8.0 and dimeric forms at pI 6.55, 6.75, 6,85, and 6.95. New bands appearing at pI 6.65 and 6.75 after treatment of sMi-CK with carboxypeptidase B were found to be delysined forms. sMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The K-m value of sMi-CK for creatine phosphate (PCr) was 1.19 +/- 0.20 mmol/L (mean +/- standard error), the activation energy (Ea) was 108.3 +/- 1.2 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 79.6 +/- 1.9%. On the other hand, octameric uMi-CK was focused at pI 7.1-7.9 and the dimeric forms were focused at pI 6.6, 6.7, 6.8, 6.9, and 7.0. Delysined forms were focused around pI 6.3, 6.4, 6.8, and 6.9. uMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pi 5.8. The K-m value of uMi-CK for PCr was 1.07 +/- 0.03 mmol/L, Ea of uMi-CK was 110.0 +/- 0.9 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 90.3 +/- 0.4%. The sMi-CK and uMi-CK were hybridized and the hybrid Mi-CK appeared at pI 6.78, 6.98, and 7.1-7.95. The pIs bf the hybrid Mi-CK were between those of sMi-CK and uMi-CK. Bs described above, sMi-CK and uMi-CK were slightly different from each other with respect to the pi and some enzyme characteristics.