Uncoupling proteins (UCP) may influence thermogenesis. Since skeletal muscle plays an important role in energy homeostasis and substrate oxidation, this study was undertaken to test the hypotheses that skeletal muscle UCP2 content is altered in obesity and could be linked to basal energy expenditure, insulin sensitivity, or substrate oxidation within skeletal muscle under postabsorptive (fasting) conditions. To examine these possibilities, limb basal energy expenditure and respiratory quotient (bRQ) were measured in 18 obese nondiabetic (Ob) and lean individuals (L). Total body fat (%) ranged from 11% to 46%. In addition, insulin‐stimulated rates of glucose disposal (Rd) were measured under euglycemic hyperinsulinemic conditions. Biopsy of vastus lateralis muscle was used to measure cytochrome c oxidase (COX) enzyme activity and UCP2 content. Whereas low muscle COX activity was found in the Ob compared to L (6.9±1.6 vs. 9.6±1.2 U/g; P<0.001), skeletal muscle UCP2 content in Ob was significantly higher than in L (48±9 vs. 33±12 arbitrary units/g; P<0.05). Moreover, UCP2 content was positively correlated with percent of total body fat (r=0.57; P<0.05) and bRQ (r=0.59; P<0.01), but not with visceral fat (r=0.17; P=0.49), basal energy expenditure (r=0.07; P=0.79) or Rd (r=−0.23; P=0.34). In summary, these results indicate that if development of obesity in humans is mediated by defective expression of UCP2 within skeletal muscle, then this effect is not observed in people with established obesity. The present study also suggests that skeletal muscle UCP2 content is not related to basal energy expenditure or insulin sensitivity in humans. However, the increased content of UCP2 within skeletal muscle in obesity appears to coincide with a reduced postabsorptive lipid utilization by muscle.—Simoneau, J.‐A., Kelley, D. E., Neverova, M., Warden, C. H. Overexpression of muscle uncoupling protein 2 content in human obesity associates with reduced skeletal muscle lipid utilization. FASEB J. 12, 1739–1745 (1998)