This chapter describes the assay method, purification, and properties of isozymes of aldehyde dehydrogenase. Two isozymes of aldehyde dehydrogenase are found in the horse liver: one cytosolic (F1) and one mitrochondrial (F2). Both enzymes oxidize a variety of aliphatic and aromatic aldehydes and show strong specificity for nicotinamide adenine dinucleotide (NAD) over nicotinamide adenine dinucleotide phosphate (NADP). The cytosolic (F1) isozyme is important for ethanol metabolism based on its extreme sensitivity to disulfiram inhibition and its lower Km for NAD. Aldehyde dehydrogenase activity is determined by monitoring the rate of nicotinamide adenine dinucleotide dehydrogenase (NADH) formation spectrophotometrically or fluorimetrically. The steps involved in the purification of the enzyme are (1) extraction, (2) ammonium sulfate fractionation, (3) CM-cellulose chromatography, (4) the separation of Fl and F2 isozymes using diethylaminoethyl (DEAE)-cellulose chromatography, and (5) final purification of F1 and F2 isozyme. The assay system of Feldman and Weiner are used during purification. All the procedures are performed below 5°C, using nitrogen-saturated buffers and a nitrogen atmosphere where possible.