Mean serum creatine kinase MB isoenzyme (CK-MB) activity in 108 trained marathon runners after competition was 98 ±66 (SD) units/L measured by a quantitative electrophoretic technique (normal, <5 units/L), or 7.9% of total CK activity. These levels in asymptomatic runners were comparable with peak serum values reported in patients during acute myocardial infarction. Elevated serum levels of CK-MB in runners can arise from skeletal muscle through exertional rhabdomyolysis, from silent injury to myocardium, or from a combined tissue source. To investigate this directly, we analyzed skeletal muscle obtained by needle biopsy for CK isoenzymes from 25 trained male marathon runners and ten sedentary male subjects. The MB isoenzyme accounted for 8.9% ± 1.3% (SD) of total CK activity per gram of total protein in the skeletal muscle of runners and 3.3% ± 0.7% (SD) in control tissue, which was significant. Total CK activity was not statistically different between the two groups. Similar relative concentrations of CK-MB in skeletal muscle (8.9%) and serum after competition (7.9%) strongly suggest that elevated serum CK-MB activity in asymptomatic runners arises from a noncardiac or skeletal muscle source.