To test the hypothesis that pyruvate dehydrogenase (PDH) is differentially regulated in specific human muscles, regulation of PDH was examined in triceps, deltoid, and vastus lateralis at rest and during intense exercise. To elicit considerable glycogen use, subjects performed 30 min of exhaustive arm cycling on two occasions and leg cycling exercise on a third day. Muscle biopsies were obtained from deltoid or triceps on the arm exercise days and from vastus lateralis on the leg cycling day. Resting PDH protein content and phosphorylation on PDH-E1 alpha sites 1 and 2 were higher (P < or = 0.05) in vastus lateralis than in triceps and deltoid as was the activity of oxidative enzymes. Net muscle glycogen utilization was similar in vastus lateralis and triceps ( approximately 50%) but less in deltoid (likely reflecting less recruitment of deltoid), while muscle lactate accumulation was approximately 55% higher (P < or = 0.05) in triceps than vastus lateralis. Exercise induced (P < or = 0.05) dephosphorylation of both PDH-E1 alpha site 1 and site 2 in all three muscles, but it was more pronounced at PDH-E1 alpha site 1 in triceps than in vastus lateralis (P < or = 0.05). The increase in activity of the active form of PDH (PDHa) after 10 min of exercise was more marked in vastus lateralis ( approximately 246%) than in triceps ( approximately 160%), but when it was related to total PDH-E1 alpha protein content, no difference was evident. In conclusion, PDH protein content seems to be related to metabolic enzyme profile, rather than myosin heavy chain composition, and less PDH capacity in triceps is a likely contributing factor to higher lactate accumulation in triceps than in vastus lateralis.