To examine the importance of phosphocreatine (PCr) degradation in maintaining power output during maximal intermittent cycling, seven healthy men completed three bouts of isokinetic cycling (30 s, 100 revolutions/min) with 4 min of rest between bouts. After bout 2, blood flow to one leg was occluded by cuffing the thigh (Cuff) during the rest period to prevent PCr resynthesis while the circulation to the other leg was intact (Cont). The cuff was then removed and bout 3 completed. Muscle biopsies were sampled from the vastus lateralis of both legs just before and immediately after bout 3. Total work produced by the Cuff and Cont legs was similar during bouts 1 (9.3 +/- 0.5 and 9.6 +/- 0.5 kJ, respectively) and 2 (8.1 +/- 0.4 and 8.3 +/- kJ, respectively). Cuffing prevented the resynthesis of PCr because pre-bout 3 contents were 20.7 +/- 8.4 and 63.0 +/- 3.3 mmol/kg dry muscle in the Cuff and Cont legs, respectively. Cuffing also resulted in significantly higher muscle levels of lactate, H+ concentration (287 +/- 26 vs. 217 +/- 15 nM), ADP, AMP, and acetyl-CoA before bout 3 but had no effect on other glycolytic intermediates, ATP, or acetylcarnitine. Total work in bout 3 was significantly reduced by 15% in the Cuff leg (5.8 +/- 0.4 vs. 6.8 +/- 0.6 kJ). PCr degradation during bout 3 was 3.1 and 47.5 mmol/kg dry muscle in the Cuff and Cont legs, respectively, and lactate accumulation was minimal in both legs. Changes in all other metabolites during bout 3 were not different between legs. The results suggest that PCr contributed approximately 15% of the total ATP provision during the third 30-s bout of maximal isokinetic cycling and that most of the ATP was provided during the initial 15 s. Muscle glycogenolysis contributed minimally to ATP provision (approximately 10-15%) during the third 30-s bout, suggesting that aerobic metabolism becomes the dominant source of ATP during this model of repeated sprinting.