Aldehyde dehydrogenase isozymes (AHD-1 and AHD-5) have been isolated in a highly purified state from extracts of mouse liver mitochondria. The enzymes have distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: AHD-1, 63,000; AHD-5, 49,000. Gel exclusion chromatography, using sephadex G-200, indicated that both isozymes are dimers, although AHD-1 may also exist as a monomeric form as well. The enzymes exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-1, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate, 1.4 mM and 0.78 mM respectively, whereas AHD-5 exhibited a low Km value with acetaldehyde of 0.2 microM. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-1, pH range 6.5-7.5; AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50% inhibition occurring 13 and 0.1 microM for the AHD-1 and AHD-5 isozyme respectively. Based upon the kinetic characteristics, it is suggested that AHD-5 may be the primary enzyme for oxidizing mitochondrial acetaldehyde during ethanol oxidation in vivo.