A sensitive radioimmunoassay method for measurement of human carbonic anhydrase III (CA III), a specific enzyme of skeletal muscle, was established. The effect of 24-h competitive running on serum CA III (S-CA III) was studied in healthy male long-distance runners. S-CA III increased gradually during the first 18 h 410-fold above the initial values. No further increase was observed during the last 6 h. On a day after the termination of the race S-CA III was not statistically different from the initial value. The activity of serum creatine kinase (S-CK) and lactate dehydrogenase (S-LDH) increased gradually through the run being elevated also 1 or 2 days afterward. The lack of increase in S-CA III during the last hours of exercise, and more rapid decrease afterward than in S-CK or S-LDH, may reflect differences in the rates of penetration through the sarcolemma and different degrees of injury in different fiber types or in the half-lives of these enzymes in serum.