Reduced metabolic rate may contribute to weight gain in leptin-deficient (ob/ob) mice; however, available studies have been criticized for referencing O2 consumption (VO2) to estimated rather than true lean body mass. To evaluate whether leptin deficiency reduces energy expenditure, four separate experiments were performed: 1) NMR spectroscopy was used to measure fat and nonfat mass, permitting VO2 to be referenced to true nonfat mass; 2) dietary manipulation was used in an attempt to eliminate differences in body weight and composition between ob/ob and C57BL/6J mice; 3) short-term effects of exogenous leptin (0.3 mg. kg-1. day-1) on VO2 were examined; and 4) body weight and composition were compared in leptin-repleted and pair-fed ob/ob animals. ob/ob animals had greater mass, less lean body mass, and a 10% higher metabolic rate when VO2 was referenced to lean mass. Dietary manipulation achieved identical body weight in ob/ob and C57BL/6J animals; however, despite weight gain in C57BL/6J animals, percent fat mass remained higher in ob/ob animals (55 vs. 30%). Exogenous leptin increased VO2 in ob/ob but not control animals. Weight loss in leptin-repleted ob/ob mice was greater than in pair-fed animals (45 vs. 17%). We conclude, on the basis of the observed increase in VO2 and accelerated weight loss seen with leptin repletion, that leptin deficiency causes a reduction in metabolic rate in ob/ob mice. In contrast, these physiological studies suggest that comparison of VO2 in obese and lean animals does not produce useful information on the contribution of leptin to metabolism.