OBJECTIVE Polycystic ovarian syndrome (PCOS) is associated with skeletal muscle insulin resistance (IR), which has been linked to decreased mitochondrial function. We measured mitochondrial respiration in lean and obese women with and without PCOS using high-resolution respirometry. METHODS Hyperinsulinemic-euglycemic clamps (40  mU/min per m(2)) and muscle biopsies were performed on 23 women with PCOS (nine lean (body mass index (BMI) <25 kg/m(2)) and 14 obese (BMI >25 kg/m(2))) and 17 age- and weight-matched controls (six lean and 11 obese). Western blotting and high-resolution respirometry was used to determine mitochondrial function. RESULTS Insulin sensitivity decreased with PCOS and increasing body weight. Mitochondrial respiration with substrates for complex I and complex I+II were similar in all groups, and PCOS was not associated with a decrease in mitochondrial content as measured by mitochondrial DNA/genomic DNA. We found no correlation between mitochondrial function and indices of insulin sensitivity. CONCLUSIONS In contrast to previous reports, we found no evidence that skeletal muscle mitochondrial respiration is reduced in skeletal muscle of women with PCOS compared with control subjects. Furthermore, mitochondrial content did not differ between our control and PCOS groups. These results question the causal relationship between reduced mitochondrial function and skeletal muscle IR in PCOS.