The effect of long chain triglyceride infusions (Intralipid 20%, 1 ml/min) on total body glucose uptake, glucose oxidation and glucose storage was examined in 25 healthy young volunteers by employing the euglycemic insulin clamp technique in combination with indirect calorimetry. Insulin was infused at three different rates (0.5, 2 and 4 mU/kg min) to achieve steady state hyperinsulinemic plateaus of 60 ± 4, 170 ± 10 and 420 ± 15 μU/ml. Prior to Intralipid infusion, the mean basal plasma free fatty acid concentration of all subjects was 385 ± 8 μmole/l. Following 90 min Intralipid infusion, the mean plasma free fatty acid level was increased to 760 ± 20 μmole/l (p < 0.001). At each insulin dose level, hyperlipidemia caused a significant reduction in total glucose uptake (5.9–3.5, 9.9–7.1, 11.1–8.8 mg/kg min, all p < 0.001. The decrease in total body glucose uptake was reflected by a decrease in both total glucose oxidation (2.4–1.6, 3.4–2.2, 3.7–2.8 mg/kg min, all p < 0.001) and glucose storage (3.6–1.9, 6.5–4.9, 7.4–5.9 mg/kg min, all p < 0.001). Basal glucose oxidation (1.3 ± 0.1 mg/kg min) fell by about 30% following 90 min of Intralipid infusion (0.9 ± 0.1 mg/kg min). Six additional subjects were studied with a lower infusion rate of Intralipid (0.5 ml/min). In these studies, insulin was infused at two different doses (0.5 and 2 mU/kg min) to achieve steady state plasma levels of 62 ± 2 and 171 ± 4 μU/ml. Intralipid caused again a significant reduction in total body glucose uptake during both the low (5.9 to 4.5 mg/kg min, p < 0.001) and the high (9.9–8.7 mg/kg min, p < 0.01) insulin clamp studies. This decrease in total glucose uptake was again the combined effect of an inhibition of both glucose storate (p < 0.05) and glucose oxidation (p < 0.001). In both high and low dose Intralipid infusion protocols, a strong inverse correlation was noted between the plasma free fatty acid concentration during the insulin clamp study and total body glucose uptake (r = 0.92, p < 0.001), glucose oxidation (r = 0.95, p < 0.001), and glucose storage (r = 0.90, p < 0.01). These results indicate that the inhibitory effect of free fatty acids on glucose utilization involves the biochemical pathways regulating both glucose oxidation and glycogen synthesis.