A sensitive enzyme immunoassay system for measurement of MM and MB forms of human creatine kinase (CK) was developed using purified monospecific antibodies to the M subunit and to the B subunit of CK. The CK-MM assay system consisted of polystyrene balls with immobilized F(ab')2 fragments of anti-M and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The CK-MB was assayed with the polystyrene balls with either antibody (anti-M or anti-B) F(ab')2 fragments and the other antibody (anti-B and anti-M, respectively) Fab' fragments labeled with galactosidase. The assays were highly sensitive and 3 pg of CK-MM and CK-MB were measurable. The CK-MM assay was specific to the M subunit of creatine kinase, and it cross-reacted about 15% with CK-MB, but not with CK-BB. The CK-MB assay did not cross-react with CK-MM nor CK-BB. Therefore, concentrations of CK-MM could be estimated by subtracting the cross-reacting values of CK-MB. Coefficients of variation in within-run and between-run precision studies for serum CK-MM and CK-MB were less than 9%. Serum levels in healthy adults of various ages (16-59 yr old) were ranged from 35.2-132 ng/ml for CK-MM and from 0.40-1.77 ng/ml for CK-MB. There was apparently no statistical significance among the sex- and age-related differences. Concentrations of CK-MM and CK-MB in various human tissues were also determined. The CK-MM was present abundantly in the heart and the tissues composed of striated muscles (skeletal muscle, diaphragm and proximal esophagus). The CK-MB was distributed not only in the heart but also in the striated muscle tissues at a relatively high level.