Serum concentrations of myoglobin (S-Myo) and carbonic anhydrase III (S-CA III; EC 184.108.40.206), a skeletal muscle-specific protein, were measured by RIA in 26 patients with acute myocardial infarction, 14 patients with neuromuscular diseases, and six healthy subjects before and after physical exercise. S-Myo was increased in infarct patients, whereas S-CA III was not altered. In patients with neuromuscular diseases and in healthy subjects after physical exercise, both S-Myo and S-CA III were significantly increased. S-CA III and S-Myo also showed identical peak times, 2 h postexercise. The S-Myo/S-CA III ratio was always higher in infarct patients than in the other groups. Thus, the combination of S-CA III and S-Myo determinations is useful to differentiate whether serum myoglobin is originating from myocardium or from skeletal muscle.