Hepatic microsomes, obtained from rats pair-fed liquid diets supplemented with either ethanol or an isocaloric amount of carbohydrates (for 4 weeks), were subjected to crossed immunoelectrophoresis. Anti-acetaldehyde adduct-specific immunoglobulin reacted on the protein blots with a single major 52,000 dalton polypeptide. This same protein was recognized by antibodies specific for P450IIE1, an ethanol-inducible P450 isozyme. Furthermore, a single protein, also reactive with anti-P450IIE1 IgG, was isolated from liver microsomes of ethanol-fed rats by immunoaffinity chromatography on Sepharose-conjugated anti-acetaldehyde adduct IgG. These results indicate that P450IIE1 is a target protein for acetaldehyde binding in liver microsomes in vivo.