Athletes in training have significantly higher levels of serum aldolase activity at rest when compared to nonathletes. This is due to the higher level (and higher proportion) of aldolase isoenzyme A, predominant in muscle. At rest, athletes with a history of infectious hepatitis show significantly higher proportional and absolute levels of aldolase B, predominant in liver. Long-lasting exercise leads to a rise in serum aldolase activity, which must be ascribed to the increase in isoenzyme A. Significant post-exercise changes in isoenzyme B were not observed. There was no correlation between changes in serum hemoglobin, as reflecting intravascular hemolysis, and changes in serum aldolase activity. The data are discussed in regard to the existing hypotheses regarding increases in serum enzyme activity after physical exercise.