Study of creatine phosphokinase (CPK) isoenzymes using a quantitative column chromatographic technique showed that there was a significant difference in serum MB isoenzyme activity between patients with Duchenne muscular dystrophy (42 mU per milliter) and those with other kinds of neuromuscular diseases (8 mU per milliter). In the Duchenne patients, the serum MB isoenzyme activity did not correlate with clinical cardiac disease. Furthermore, skeletal muscle damage produced in the rat by aortic ligation and 5-hydroxytryptamine resulted in significant elevations of plasma MB isoenzyme activty. These studies suggest that the serum MB isoenzyme activity in the Duchenne muscular dystrophy patients is probably arising from skeletal, not cardiac muscle and may reflect a distinct pathogenesis from other kinds of neuromuscular disorders.