BACKGROUND Although the clinical manifestations of alcoholic liver disease are well described, little is known about the molecular basis for liver injury. Recent studies have indicated that chronic alcohol consumption leads to the lysine-hyperacetylation of several hepatic proteins, and this list is growing quickly. METHODS To identify other hyperacetylated proteins in ethanol-fed livers, we chose a proteomics approach. Cytosolic and membrane proteins (excluding nuclei) were separated on 2D gels, transferred to PVDF and immunoblotted with antibodies specific for acetylated lysine residues. Hyperacetylated proteins were selected for trypsin digestion and mass spectrometric analysis. RESULTS In all, 40 proteins were identified, 11 of which are known acetylated proteins. Remarkably, the vast majority of hyperacetylated membrane proteins were mitochondrial residents. Hyperacetylated cytosolic proteins ranged in function from metabolism to cytoskeletal support. Notably, 3 key anti-oxidant proteins were identified whose activities are impaired in ethanol-treated cells. We confirmed that the anti-oxidant enzyme, glutathione peroxidase 1, actin and cortactin are hyperacetylated in ethanol-treated livers. CONCLUSIONS Alcohol-induced hyperacetylation of multiple proteins may contribute to the development of liver injury. The abundance of acetylated mitochondrial proteins further suggests that this modification is important in regulating liver metabolism and when perturbed, may contribute to the progression of a variety of metabolic diseases.