The subcellular distribution and relative amounts of the two isozymes, F1 and F2, of aldehyde dehydrogenase (EC 220.127.116.11) which were recently purified to homogeneity from horse liver (Eckfeldt, J., et al. (1976) J. Biol. Chem. 251, 236-240) have been investigated. A fresh horse liver homogenate was fractionated on DEAE-cellulose. The results indicate that approximately 60% of the total pH 7.0 acetaldehyde dehydrogenase activity is due to the F1 isozyme and 40% is due to the F2 isozyme. Several horse livers were then fractionated into subcellular components using a differential centrifugation method. Based on the disulfiram (Antabuse) inhibition and the aldehyde concentration dependence of the enzymatic activity, it appears that the disulfiram-sensitive F1 isozyme (Km acetaldehyde ≅ 70 μm) is primarily cytosolic and the disulfiram-insensitive F2 isozyme (Km acetaldehyde ≅ 0.2 μm) is primarily mitochondrial. Fluorescence studies showed that the acetaldehyde dehydrogenase of the intact mitochondria could utilize only the endogenous pyridine nucleotide pool and not externally added NAD. Also, the ethanol dehydrogenase activity was found to be nearly 10 times the total acetaldehyde dehydrogenase activity when assaying a horse liver homogenate at pH 7.0 and with saturating substrates. The significant differences between this work and the results reported in rat liver are discussed with respect to the physiological importance of the cytosolic and mitochondrial aldehyde dehydrogenase during the ethanol oxidation in vivo.