The proportion of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB activity was increased in skeletal muscle biopsies obtained from five long-distance runners, both 2 h before (mean 7.7%, SD 2.4%) and 30 min after (mean 7.2%, SD 1.2%) a marathon race, as compared with that in biopsies from five nonrunners (controls less than or equal to 1.0%). Further, mitochondrial CK and CK-BB isoenzymes were present in homogenates of the runners' skeletal muscle samples but not in those of the nonrunners. However, there were no substantial differences in the mean total CK activities per gram (wet wt.) of muscle tissue among premarathon samples, postmarathon samples, and nonrunners' samples (3148, 3365, and 3049 U/g, respectively). We conclude that the metabolically active gastrocnemius muscle of long-distance runners is qualitatively similar to the heart muscle in its CK isoenzyme composition.