Acetaldehyde, the first metabolite of ethanol, has been shown to be capable of binding covalently to liver proteins in vivo, which may be responsible for a variety of toxic effects of ethanol. Acetaldehyde-protein adducts have previously been detected in the liver of patients and experimental animals with alcoholic liver disease. Although a role for acetaldehyde as a possible mediator of ethanol-induced neurotoxicity has also been previously suggested, the formation of protein-acetaldehyde adducts in brain has not been examined. This study was designed to examine the occurrence of acetaldehyde-protein adducts in rat brain after lifelong ethanol exposure. A total of 27 male rats from the alcohol-preferring (AA) and alcohol-avoiding (ANA) lines were used. Four ANA rats and five AA rats were fed 10-12% (v/v) ethanol for 21 months. Both young (n = 10) and old (n = 8) rats receiving water were used as controls. Samples from frontal cortex, cerebellum and liver were processed for immunohistochemical detection of acetaldehyde adducts. In four (two ANA, two AA rats) of the nine ethanol-exposed rats, weak or moderate positive reactions for acetaldehyde adducts could be detected both in the frontal cortex and cerebellum, whereas no such immunostaining was found in the remaining five ethanol-treated rats or in the control rats. The positive reaction was localized to the white matter and some large neurons in layers 4 and 5 of the frontal cortex, and to the molecular layer of the cerebellum. Interestingly, the strongest positive reactions were found among the ANA rats, which are known to display high acetaldehyde levels during ethanol oxidation. We suggest that acetaldehyde may be involved in ethanol-induced neurotoxicity in vivo through formation of adducts with brain proteins and macromolecules.