Experimentally induced hypertriglyceridemia (HTG) and high plasma free fatty acid (FFA) levels impair in vivo insulin action. To determine if this is a consequence of impaired in vivo insulin receptor autophosphorylation and related to defective receptor signaling, hyperinsulinemic euglycemic clamps, indirect calorimetry, and skeletal muscle biopsies were performed in nine healthy subjects. In vivo insulin action was determined from the glucose infusion rate (GINF) and glucose oxidation (Glcox) during 40 and 120 mU/m2 /min clamps with (HTG clamp) and without (control clamp) a triglyceride emulsion infusion. The percentage of receptors autophosphorylated in vivo was determined by 125I-labeled insulin tracer binding in skeletal muscle immunoprecipitates of insulin receptors and phosphorylated receptors. Compared with the control clamps, plasma triglycerides and FFA increased four- and twofold, whereas GINF and Glcox decreased 15 and 35%, respectively, during the HTG clamps (all P<0.05). However, the percentages of receptors phosphorylated after the 40 and 120 mU/m2/min HTG clamps (9.2 +/- 1.5 and 21.1 +/- 2.6%, respectively) were similar to the control clamps (9.0 +/- 0.6 and 18.6 +/- 2.2%, respectively). These results indicate that, if impaired insulin signal transduction is a mechanism by which HTG and FFA impair insulin action, it occurs at a site downstream from insulin receptor autophosphorylation.