Ethanol-induced damage is largely attributed to its toxic metabolite, acetaldehyde. Clearance of acetaldehyde is achieved by its oxidation, primarily catalyzed by the mitochondrial class II aldehyde dehydrogenase (ALDH2). ALDH1B1 is another mitochondrial aldehyde dehydrogenase (ALDH) that shares 75% peptide sequence homology with ALDH2. Recent population studies in whites suggest a role for ALDH1B1 in ethanol metabolism. However, to date, no formal documentation of the biochemical properties of ALDH1B1 has been forthcoming. In this current study, we cloned and expressed human recombinant ALDH1B1 in Sf9 insect cells. The resultant enzyme was purified by affinity chromatography to homogeneity. The kinetic properties of purified human ALDH1B1 were assessed using a wide range of aldehyde substrates. Human ALDH1B1 had an exclusive preference for NAD(+) as the cofactor and was catalytically active toward short- and medium-chain aliphatic aldehydes, aromatic aldehydes, and the products of lipid peroxidation, 4-hydroxynonenal and malondialdehyde. Most importantly, human ALDH1B1 exhibited an apparent K(m) of 55 μM for acetaldehyde, making it the second low K(m) ALDH for metabolism of this substrate. The dehydrogenase activity of ALDH1B1 was sensitive to disulfiram inhibition, a feature also shared with ALDH2. The tissue distribution of ALDH1B1 in C57BL/6J mice and humans was examined by quantitative polymerase chain reaction, Western blotting, and immunohistochemical analysis. The highest expression occurred in the liver, followed by the intestinal tract, implying a potential physiological role for ALDH1B1 in these tissues. The current study is the first report on the expression, purification, and biochemical characterization of human ALDH1B1 protein.