Rates of NADPH generation by the pentose phosphate pathway were evaluated in perfused livers from ethanol-fed or control rats by measuring the production of 14CO2 from 1-14C-glucose. Under basal perfusion conditions, livers from ethanol-fed rats released lactate and pyruvate into the perfusate at rates that were only 19% of the control values. Under these conditions, calculated rates of NADPH generation by the pentose cycle in livers of the ethanol-fed rats were only 50% of rates obtained with livers of control rats. 7-Ethoxycoumarin (7-EC), a substrate for mixed function oxidation, was infused to increase rates of hepatic NADPH utilization. In livers from control rats, 7-EC was oxidized at a rate of 2.6 mumol/g/hr, but rates of NADPH generation by the pentose cycle were increased by 8.8 mumol/g/hr. In livers from ethanol-fed rats, 7-EC was metabolized at rates of 7.2 mumol/g/hr, but the generation of NADPH by the pentose cycle was increased by only 3.9 mumol/g/hr. The infusion of 7-EC was associated with increases in rates of O2 uptake that exceeded rates of mixed function oxidation in both groups of animals. Ethanol feeding decreased the activity of glucose-6-phosphate dehydrogenase by 40% and decreased the concentrations of glycogen by 66%. Thus, the decrease in pentose cycle flux in perfused livers may be due to diminished activity of the rate-controlling enzyme and/or diminished substrate supply from glycogen. However, cytosolic NADP+/NADPH ratios were identical in livers of both groups. Because NADPH was not depleted during the mixed function oxidation of 7-EC in livers from ethanol-fed rats, it is concluded that other hepatic sources of NADPH compensate for the diminished generation by the pentose cycle.