1. The activity of pyruvate dehydrogenase in its active and inactive forms was measured in biopsy samples obtained from the vastus lateralis muscle of healthy subjects before and after exercise. 2. At rest, 40 +/- 4% (mean +/- SEM) of the enzyme was in the active form. 3. After progressive aerobic exercise to exhaustion (n = 5), 88 +/- 2.3% was in the active form. 4. After intermittent supramaximal short-term exercise (1 min exercise, 3 min rest) to exhaustion (n = 6), 60 +/- 2.2% was in the active form. 5. After isometric maximal exercise of 65 +/- 3.6 s duration (n = 3), only 39 +/- 1% of the enzyme was in the active form. 6. Muscle glycogen depletion was greatest with intermittent exercise and least with isometric maximal exercise; in contrast, the increase in muscle lactate was least with progressive exercise (1.3 to 9.4 mumol/g), intermediate in intermittent maximal exercise (1.2 to 13.1 mumol/g) and greatest after isometric exercise (1.8 to 17.6 mumol/g). There were no significant differences between the three studies in the changes in lactate/pyruvate ratios. 7. In three subjects who exercised with one leg, activation of the enzyme was twice as great in the exercise as in the inactive leg. 8. The ratio of active to total enzyme in biopsies of resting muscle was greater in four well-trained athletes than in four untrained control subjects (70% compared with 41% respectively). 9. The activation of pyruvate dehydrogenase appears to play an important part in regulating the use of glycogen and glucose during exercise in man.