5'-AMP-activated protein kinase (AMPK) was recently suggested to regulate pyruvate dehydrogenase (PDH) activity and thus pyruvate entry into the mitochondrion. We aimed to provide evidence for a direct link between AMPK and PDH in resting and metabolically challenged (exercised) skeletal muscle. Compared with rest, treadmill running increased AMPKalpha1 activity in alpha(2)KO mice (90%, P < 0.01) and increased AMPKalpha2 activity in wild-type (WT) mice (110%, P < 0.05), leading to increased AMPKalpha Thr(172) (WT: 40%, alpha(2)KO: 100%, P < 0.01) and ACCbeta Ser(227) phosphorylation (WT: 70%, alpha(2)KO: 210%, P < 0.01). Compared with rest, exercise significantly induced PDH-E(1)alpha site 1 (WT: 20%, alpha(2)KO: 62%, P < 0.01) and site 2 (only alpha(2)KO: 83%, P < 0.01) dephosphorylation and PDH(a) [ approximately 200% in both genotypes (P < 0.01)]. Compared with WT, PDH dephosphorylation and activation was markedly enhanced in the alpha(2)KO mice both at rest and during exercise. The increased PDH(a) activity during exercise was associated with elevated glycolytic flux, and muscles from the alpha(2)KO mice displayed marked lactate accumulation and deranged energy homeostasis. Whereas mitochondrial DNA content was normal, the expression of several mitochondrial proteins was significantly decreased in muscle of alpha(2)KO mice. In isolated resting EDL muscles, activation of AMPK signaling by AICAR did not change PDH-E(1)alpha phosphorylation in either genotype. PDH is activated in mouse skeletal muscle in response to exercise and is independent of AMPKalpha2 expression. During exercise, alpha(2)KO muscles display deranged energy homeostasis despite enhanced glycolytic flux and PDH(a) activity. This may be linked to decreased mitochondrial oxidative capacity.