In vitro studies indicating an inverse relationship between free fatty acid (FFA) availability and glucose oxidation led to proposal of the glucose-fatty acid cycle. In vivo studies have yielded conflicting results regarding the effect of FFA on glucose oxidation. In the present study the effect of FFA on glucose oxidation was determined in six normal volunteer subjects. The rate of glucose uptake was fixed by using a constant glucose infusion to suppress endogenous glucose production. Glucose was infused continuously overnight and during the four hour study at 8 mg/kg × min to ensure use of glucose as an energy substrate by virtually all tissues. Following a two-hour baseline glucose infusion, infusion of 20% IV fat emulsion at 1.0 mL/min plus heparin was added to the constant glucose infusion for two additonal hours. Total carbohydrate oxidation was determined by indirect calorimetry, and the direct oxidation of the infused (plasma) glucose was measured by the use of U-¹³C-glucose. Glycogen oxidation was calculated as the difference between total carbohydrate oxidation and the oxidation of plasma glucose. Glucose uptake was calculated from the infusion rate, corrected for any changes in plasma and/or urine glucose concentration. Glucose uptake closely approximated the rate of IV glucose infusion and was unchanged by fat infusion. The percent of CO₂ production from U-¹³C-glucose oxidation (74.5 ± 12.3, mean ± SD) was not affected by FFA, nor was the percent of glucose uptake oxidized (37.5 ± 4.0). Indirect calorimetry showed a decrease of total carbohydrate oxidation from 4.22 ± 1.12 mg/kg × min to 3.13 ± 1.65 mg/kg × min (P < .05) during the final hour of fat infusion. The calculated value for glycogen oxidation (the oxidation of glucose which never entered the plasma pool) significantly decreased with increased FFA availability (0.97 ± 1.18 to 0.05 ± 1.54 mg/kg × min) (P < .05). These data suggest that FFAs do not inhibit plasma glucose oxidation when the rate of glucose uptake is constant. Increases in circulating FFAs may affect total glucose oxidation, however, by suppressing the oxidation of glycogen.