Sirt1 is an NAD+-dependent histone deacetylase which interacts with the regulatory protein of mitochondrial biogenesis PGC-1, and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins and PGC-1 existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days 10 Hz; 3 hrs/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1 levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, while Sirt1 activity remained unchanged. In chronically-stimulated muscle, cytochrome c was increased by 30% compared to non-stimulated muscle, while Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle these markers of mitochondrial content were decreased by 30-50% compared to the control muscle, while Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1 expression are independently regulated and that while Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.