Free radical products have previously been detected in rodents after chronic feeding with an ethanol-containing, high-fat diet. The significance of reactive free radical formation in ethanol-induced hepatotoxicity has been difficult to assess because most rodent models exhibit only fatty liver. However, serious hepatic damage resembling clinical alcoholic liver injury (e.g., steatosis, inflammation, and necrosis) occurs in rats after continuous intragastric administration of an ethanol-containing, high-fat diet developed by Tsukamoto and French. Accordingly, rats treated with ethanol for at least 2 weeks using this protocol were administered the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and bile samples were collected. A six-line radical adduct spectrum was detected in the bile of ethanol-treated rats. A similar spectrum of lower intensity was detected with rats fed a high-fat diet without ethanol, but little or no radical adduct signal was detected with chow-fed animals. For both treatment groups, alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and extra ethanol were given acutely. Destruction of Kupffer cells by chronic treatment with GdCl3 decreased by about 50% the radical adduct formation in rats fed the ethanol-containing, high-fat diet. This radical species was largely ethanol derived, because addition of [13C]ethanol produced a 12-line spectrum, indicating the formation of alpha-hydroxyethyl radical. Ethanol treatment also caused hypoxia (detected on the liver surface in vivo with oxygen electrodes), which was reflected in a dose-dependent decrease in oxygen tension with ethanol. The effect was blocked by GdCl3. Hepatic damage detected by histology was prevalent in ethanol-treated rats but only mild fatty liver was observed in high-fat diet-fed controls. GdCl3 treatment eliminated hepatic damage due to high-fat and ethanol diets, and when all groups were compared a significant correlation between liver injury and radical adduct signal was observed. Thus, free radical formation in ethanol-treated rats has been detected for the first time in a model that exhibits injury characteristic of human alcoholic injury, and signal intensity correlates with hepatotoxicity. Moreover, the decrease in both free radical formation and hepatic damage produced by GdCl3 implicates Kupffer cells in the development of alcoholic liver injury. This important pathophysiological process may involve direct production of reactive oxygen species or indirect actions of mediators on parenchymal cells.