We sought to ascertain whether pretreatment with troglitazone (20 days) could prevent acute free fatty acid (FFA)–induced insulin resistance in male Wistar rats. Animals were divided into three groups: 1) control, 2) FFA infusion alone (FFA1), and 3) thiazolidinedione (TZD)-treated + FFA infusion (FFA1). Days before a hyperinsulinemic-euglycemic clamp, all animals were cannulated in the jugular vein (infusion) and carotid artery (sampling). Animals were allowed 5 days to recover from surgery and fasted 12 h before the experiment. Glucose (variable), insulin (40 mU · kg⁻¹ · min⁻¹), and Liposyn (heparinized 10% lipid emulsion) infusions were initiated simultaneously and continued from 0–120 min. Steady-state glucose, 8.3 ± 0.14 mmol/l, and insulin concentrations, 7.3 ± 2.45 nmol/l, were the same between groups. Interestingly, steady-state FFA levels were significantly lower in animals pretreated with TZD compared with FFA alone (1.83 ± 0.26 vs. 2.96 ± 0.25 mmol/l; P = 0.009), despite matched intralipid infusion rates. A second group of TZD-treated animals (TZD + FFA2) were infused with intralipid at a higher infusion rate (44%) to match the arterial concentrations of FFA1. The glucose infusion and insulin-stimulated glucose disposal rates (GDRs) were significantly decreased (40%) for untreated Liposyn infused (FFA1) compared with control rats. In addition, insulin receptor substrate-1 (IRS-1) phosphorylation and IRS-1–associated phosphatidylinositol (PI) 3-kinase activity was significantly reduced, 30–50%, in FFA1 rats. TZD pretreatment prevented the FFA-induced decrement in insulin signaling. Fatty acid translocase (FAT/CD36) also was significantly reduced (56%) in untreated FFA1 rats after the clamp but remained identical to control values for TZD-treated rats. In conclusion, acutely elevated FFA levels 1) induced a significant reduction in tracer-determined GDR paralleled by impaired tyrosine phosphorylation of IRS-1 and reduced IRS-1–associated PI 3-kinase activity and 2) induced a significant reduction in FAT/CD36 total protein. TZD pretreatment prevented FFA-induced decrements in insulin action and prevented the reduction in FAT/CD36 protein.