Aldehyde dehydrogenase was partially purified from human liver. During purification, activity was resolved into one major and one minor species by DEAE-cellulose column chromatography; the properties of the predominant form were investigated.Aldehydes are oxidized when NAD+, but not NADP+, is the electron acceptor, maximal activity occurring between pH 9 and 10. Several aliphatic aldehydes and hydroxyaldehydes served as substrates for the enzyme. Benzaldehyde also was oxidized, but at a comparatively low rate. Aliphatic aldehydes carrying negatively charged groups are not oxidized. The enzyme is sensitive to low concentrations of two sulfhydryl reagents, p-chloromercuribenzoate and mercuric ions; this inhibition was reversed with sulfhydryl compounds. Like other aldehyde dehydrogenases, the human liver enzyme is inhibited by arsenite and the inhibition is potentiated by mercaptoethanol. Only 35% inhibition was produced by disulfiram at 40 μM; and diethyldithiocarbamate, its metabolic reduction product, had no effect on activity below 10 mM.