Rates of 7-ethoxycoumarin (7EC) O-deethylation in perfused livers were increased approximately 3-fold by chronic ethanol feeding. The acute addition of ethanol (5 mM) and antimycin A (0.03 mM) strongly inhibited 7EC metabolism in perfused livers from ethanol-fed rats, but less inhibition was observed when these agents were added to microsomes or to perfused livers from control rats. The activity of the hepatic pentose phosphate cycle in perfused livers was assessed by comparing 14CO2 release during the infusion of 1-14C-glucose or 6-14C-glucose. 7EC infusion caused a 3-fold greater increase in 14CO2 production from 1-14C-glucose in a liver from a control rat than in a liver from an ethanol-fed rat, indicating greater hepatic pentose cycle activity in livers of control rats. Thus, the pronounced inhibition of 7EC metabolism caused by infusion of ethanol and antimycin A may be explained by a greater dependency on mitochondrial sources of NADPH in livers of ethanol-fed rats. Dinitrophenol (0.05 mM) did not inhibit 7EC metabolism in perfused livers, indicating that a reduction in the cellular redox state, and not diminished energetics, is involved in the mechanism of inhibition produced by antimycin A.