The underlying mechanism by which skeletal muscle adapts to exercise training or chronic energy deprivation is largely unknown. To examine this question, rats were fed for 9 wk either with or without β-guanadinopropionic acid (β-GPA; 1% enriched diet), a creatine analog that is known to induce muscle adaptations similar to those induced by exercise training. Muscle phosphocreatine, ATP, and ATP/AMP ratios were all markedly decreased and led to the activation of AMP-activated protein kinase (AMPK) in the β-GPA-fed rats compared with control rats. Under these conditions, nuclear respiratory factor-1 (NRF-1) binding activity, measured using a cDNA probe containing a sequence encoding for the promoter of δ-aminolevulinate (ALA) synthase, was increased by about eightfold in the muscle of β-GPA-fed rats compared with the control group. Concomitantly, muscle ALA synthase mRNA and cytochrome ccontent were also increased. Mitochondrial density in both extensor digitorum longus and epitrochlearis from β-GPA-fed rats was also increased by more than twofold compared with the control group. In conclusion, chronic phosphocreatine depletion during β-GPA supplementation led to the activation of muscle AMPK that was associated with increased NRF-1 binding activity, increased cytochromec content, and increased muscle mitochondrial density. Our data suggest that AMPK may play an important role in muscle adaptations to chronic energy stress and that it promotes mitochondrial biogenesis and expression of respiratory proteins through activation of NRF-1.