The localization of different classes of alcohol dehydrogenases (ADH) in the brain is of great interest because of their role in both ethanol and retinoic acid metabolism. Conflicting data have been reported in the literature. By Northern blot and enzyme activity analyses only class III ADH has been detected in adult brain specimens, while results from riboprobe in situ hybridization indicate class I as well as class IV ADH expression in different regions of the rat brain. Here we have studied the expression patterns of three ADH classes in adult rat, mouse and human tissues using radioactive oligonucleotide in situ hybridization. Specificity of probes was tested on liver and stomach control tissue, as well as tissue from class IV ADH knock-out mice. Only class III ADH mRNA was found to be expressed in brain tissue of all three investigated species. Particularly high expression levels were found in neurons of the red nucleus in human tissue, while cortical neurons, pyramidal and granule cells of the hippocampus and dopamine neurons of substantia nigra showed moderate expression levels. Purkinje cells of cerebellum were positive for class III ADH mRNA in all species investigated, whereas granular layer neurons were positive only in rodents. The choroid plexus was highly positive for class III ADH, while no specific signal for class I or class IV ADH was detected. Our results thus support the notion that the only ADH expressed in adult mouse, rat and human brain is class III ADH.