Because muscle triacylglycerol (TAG) accumulation might contribute to insulin resistance in leptin-deficient ob/obmice, we studied the acute (60- to 90-min) effects of leptin and insulin on [¹⁴C]glucose and [¹⁴C]oleate metabolism in muscles isolated from lean and obeseob/ob mice. Inob/ob soleus, leptin decreased glycogen synthesis 36–46% (P < 0.05), increased oleate oxidation 26% (P < 0.05), decreased oleate incorporation into TAG 32% (P < 0.05), and decreased the oleate partitioning ratio (oleate partitioned into TAG/CO₂) 44% (P < 0.05). Insulin decreased oleate oxidation 31% (P < 0.05), increased oleate incorporation into TAG 46% (P< 0.05), and increased the partitioning ratio 125% (P < 0.01). Adding leptin diminished insulin’s antioxidative, lipogenic effects. In soleus from lean mice, insulin increased the partitioning ratio 142%, whereas leptin decreased it 51%, as previously reported (Muoio, D. M., G. L. Dohm, F. T. Fiedorek, E. B. Tapscott, and R. A. Coleman.Diabetes 46: 1360–1363, 1997). The phosphatidylinositol 3-kinase inhibitor wortmannin blocked insulin’s effects on lipid metabolism but only attenuated leptin’s effects. Increasing glucose concentration from 5 to 10 mM did not affect TAG synthesis, suggesting that insulin-induced lipogenesis is independent of increased glucose uptake. These data indicate that leptin opposes insulin’s promotion of TAG accumulation in lean andob/ob muscles. Because acute leptin exposure does not correct insulin resistance inob/ob muscles, in vivo improvements in glucose homeostasis appear to require other long-term factors, possibly TAG depletion.