Activity assay and isoelectric focusing analysis of human biopsy and autopsy liver specimens showed the existence of two major aldehyde dehydrogenases (ALDH I, ALDH II). Subcellular distribution of these isozymes was determined in autopsy livers from alcoholics and nonalcoholics. Nearly 70% of the total ALDH activity was recovered in the cytosol which contained both the major isozymes. Densitometric evaluation of isozyme bands showed that about 65% of the cytosolic enzyme activity was due to ALDH II and the rest due to ALDH I isozyme. Only about 5% of the total ALDH activity was found in the mitochondrial fraction (70% ALDH I and 30% ALDH II). Significantly reduced total and specific ALDH activities were noted in all the subcellular fractions from cirrhotic liver specimens. Apparently, ALDH I isozyme from cytosol and mitochondria is primarily responsible for the oxidation of small amounts of acetaldehyde normally found in the blood of nonalcoholics after drinking moderate amounts of alcohol. However, in alcoholics who exhibit higher blood acetaldehyde concentrations after drinking alcohol, ALDH II isozyme may be of greater physiological significance.