A close relationship between elevated plasma free fatty acid (FFA) levels and insulin resistance is commonly reported in obese subjects. The aim of the present study was to evaluate the role of intramuscular triglyceride (mTG) and FFA levels in insulin sensitivity in 30 nondiabetic normal-weight or obese subjects (18 with body mass index [BMI] = 21.8 ± 3.3 kg/m² and 12 with BMI = 34.6 ± 2.7 kg/m²) who underwent minor abdominal surgery. Body composition was estimated by isotopic dilution, substrate oxidation by indirect calorimetry, and whole-body glucose uptake by euglycemic-hyperinsulinemic clamp (EHC). Glucose uptake (M) value negatively correlated with the MTG level (R² = −.56, P < .0001), which was increased in obese different in the 2 groups: an increased concentration of saturated fat was present in obese patients (unsaturated to saturated ratio, 1.89 ± 0.40 v 2.19 ± 0.07, P < .0001). Stepwise linear regression analysis of total mTGs and palmitic and oleic fractions on the M value showed that only TGs and plamitic acid were significantly related to glucose uptake (R² = .66, P < .0001). Furthermore, among the other anthropometric variables, only the BMI was significantly correlated with MTGs (R² = .71, P < .0001). In conclusion, not only the MTG concentration but also the FFA pattern seems to affect insulin-mediated glucose uptake. A pivotal role might be played by a high saturated fatty acid content in the TGs.