Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll-like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol-induced liver injury, the long-term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild-type mice (C3H/HeOuJ) were compared. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver-to-body weight ratios and serum alanine transaminase (ALT) levels in wild-type mice (109 Ψ 18 U/L) over high-fat controls (40 Ψ 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 Ψ 9 U/L). While no significant pathologic changes were observed in high-fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals (pathology score β«Ψβ¬ 5.2 Ψ 1.2). These pathologic changes were significantly lower in TLR4-deficient mice fed ethanol (score β«Ψβ¬ 2.0 Ψ 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor β£ (TNF-β£) mRNA expression in wildtype, but not in TLR4-deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol-induced liver injury.