Deacetylation of PGC‐1α by SIRT1 is thought to be an important step in increasing PGC‐1α transcriptional activity, since in muscle cell lines SIRT1 induces PGC‐1α protein expression and mitochondrial biogenesis. We examined the relationship between SIRT1 protein and activity, PGC‐1α and markers of mitochondrial density, (a) across a range of metabolically heterogeneous skeletal muscles and the heart, and when mitochondrial biogenesis was stimulated by (b) chronic muscle stimulation (7 days) and (c) AICAR administration (5 days), and finally, (d) we also examined the effects of SIRT1 overexpression on mitochondrial biogenesis and PGC‐1α. SIRT1 protein and activity were correlated (r= 0.97). There were negative correlations between SIRT1 protein and PGC‐1α (r=−0.95), COX IV (r=−0.94) and citrate synthase (r=−0.97). Chronic muscle stimulation and AICAR upregulated PGC‐1α protein (22–159%) and oxidative capacity (COX IV, 20–69%); in each instance SIRT1 protein was downregulated by 20–40%, while SIRT1 intrinsic activity was increased. SIRT1 overexpression in rodent muscle increased SIRT1 protein (+240%) and doubled SIRT1 activity, but PGC‐1α (−25%), mtTFA (−14%) and COX IV (−10%) proteins were downregulated. Taken altogether these experiments are not consistent with the notion that SIRT1 protein plays an obligatory regulatory role in the process of PGC‐1α‐mediated mitochondrial biogenesis in mammalian muscle.