Retinol dehydrogenase 13 (RDH13) is a recently identified short-chain dehydrogenase/reductase related to microsomal retinoid oxidoreductase RDH11. In this study, we examined the distribution of RDH13 in human tissues, determined its subcellular localization and characterized the substrate and cofactor specificity of purified RDH13 in order to better understand its properties. The results of this study demonstrate that RDH13 exhibits a wide tissue distribution and, by contrast with other members of the RDH11-like group of short-chain dehydrogenases/reductases, is a mitochondrial rather than a microsomal protein. Protease protection assays suggest that RDH13 is localized on the outer side of the inner mitochondrial membrane. Kinetic analysis of the purified protein shows that RDH13 is catalytically active and recognizes retinoids as substrates. Similar to the microsomal RDHs, RDH11, RDH12 and RDH14, RDH13 exhibits a much lower Km value for NADPH than for NADH and has a greater catalytic efficiency in the reductive than in the oxidative direction. The localization of RDH13 at the entrance to the mitochondrial matrix suggests that it may function to protect mitochondria against oxidative stress associated with the highly reactive retinaldehyde produced from dietary beta-carotene.