The endoplasmic reticulum (ER) contains a number of thiol-disulfide oxidoreductases of the protein disulfide isomerase (PDI)-family that catalyze the formation of disulfide bonds in newly synthesized proteins. Here we describe the identification and characterization of a novel member of the human PDI-family, the thioredoxin-related transmembrane protein 3 (TMX3). The TMX3 gene encodes a protein of 454 amino acid residues that contains a predicted N-terminal signal sequence, a single domain with sequence similarity to thioredoxin and a CGHC active-site sequence, a potential transmembrane domain and a C-terminal KKKD tetrapeptide sequence that matches the classical KKXX-type consensus sequence for ER retrieval of type I transmembrane proteins. Endogenous TMX3 contains endoglycosidase H sensitive glycans, localizes to the ER by immunofluorescence microscopy and is present in the membrane fraction after alkaline extraction of the ER lumenal content. The TMX3 transcript is found in a variety of tissues and is not upregulated by the unfolded protein response. Circular dichroism spectroscopy of the recombinantly expressed lumenal domain of TMX3 showed features typical of a properly folded protein of the alpha/beta-type. The redox potential of recombinant lumenal TMX3 was determined to -0.157 V, similar to the values previously found for PDI and ERp57. Interestingly, TMX3 showed oxidase activity, and in human tissue-culture cells the protein was found partially in the oxidized form, potentially suggesting a function of the protein as a dithiol oxidase.