Although changes in the acetaldehyde (Ac-CHO) oxidizing system in the liver are important for understanding the pathogenesis of alcoholic liver injury, interspecies differences of hepatic aldehyde dehydrogenase (ALDH: 1.2.1.3) isozymes have not yet been sufficiently studied. In the present study, the character and subcellular distribution of hepatic ALDH isozymes in male animals such as the Rhesus monkey, domestic cow, albino rabbit and Wistar strain rat were analyzed and compared with those in humans. The optimal pH for ALDH isozymes in human liver was 9.5, while those of monkey, cow, rabbit and rat were 9.0, 9.0, 8.5 and 8.5, respectively. In human liver, low Km ALDH activity was distributed mainly in the cytosol, while the corresponding activity was selectively distributed in the mitochondria in rat liver. The distribution patterns of low Km ALDH in the other animals were similar to those of the rat. In microsomes, low Km ALDH activity was very low or almost negligible in the livers of all species. These results indicate that Ac-CHO degrades mainly in the cytosol in the human liver, whereas, in the other species, it occurs in the mitochondria. This suggests that results obtained with experimental animals cannot be applied directly to humans. It is also suggested that degradation of the Ac-CHO produced in the microsomes may be slow in all species.