The protein superfamily of short-chain dehydrogenases/reductases (SDRs) today comprises over 20,000 members found in pro- and eukaryotes. Despite low amino acid sequence identity (only 15–30%), they share several similar characteristics in conformational structures, the N-terminal cofactor (NAD(P)/NAD(P)H) binding region being the most conserved. The enzymes catalyze oxido-reductive reactions and have a broad spectrum of substrates. Not all recently identified SDRs have been analyzed in detail yet, and we therefore characterized two rudimentarily annotated human SDR candidates: an orphan SDR (SDR-O) and hydroxysteroid dehydrogenase like 2 (HSDL2). We analyzed the amino acid sequence for cofactor preference, performed subcellular localization studies, and a screening for substrates of the enzymes, including steroid hormones and retinoids. None of both tested proteins showed a significant conversion of steroid hormones. However, the peroxisomal localization of human HSDL2 may suggest an involvement in fatty acid metabolism. For SDR-O a weak conversion of retinal into retinol was detectable in the presence of the cofactor NADH.