Chronic ethanol feeding increases hepatic turnover and sinusoidal efflux of glutathione in rats. The present study was performed to determine whether the observed increase in glutathione efflux was due to increased extrahepatic requirements for glutathione. The concentration and disposition of plasma glutathione were determined in rats fed liquid diets containing 36% of calories as ethanol or pair-fed an isocaloric mixture with carbohydrate replacing ethanol calories for 5 to 8 weeks. The half-life and plasma clearance of [35S]glutathione were found to be similar in ethanol-fed and control rats and in rats withdrawn 24 hr from ethanol. Uptakes of the sulfur moiety of [35S]glutathione by kidney, jejunal mucosa, liver, lung, spleen, muscle and heart were also unchanged by ethanol feeding. The plasma glutathione concentration was significantly higher in ethanol-withdrawn rats 22.30 ± 3.06 nmoles per ml (p < 0.05) compared to pair-fed controls (13.51 ± 2.04), while rats continuing to drink ethanol had intermediate levels (16.96 ± 2.22). Plasma cysteine levels were slightly, but not significantly, higher in ethanol-fed rats. These findings suggest that increased sinusoidal efflux of glutathione in ethanol-fed rats is due to a direct effect of ethanol on hepatic glutathione transport and not due to an alteration in extrahepatic disposition of glutathione. In order to characterize further the effects of ethanol feeding on glutathione-dependent detoxification, activities of glutathione S-transferase, glutathione reductase and γ-glu-tamyltransferase were determined. Both glutathione S-transferase and glutathione reductase activities were significantly increased by ethanol feeding whereas γ-glutamyltransferase was increased only slightly. Thus, chronic ethanol feeding in rats results in altered hepatic glutathione metabolism, both through nondetoxification pathways and potentially by glutathione-dependent detoxification enzymes.