Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed ERO for ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000) J. Biol. Chem. 275, 4827-4833). Here, we report the cloning and characterization of a novel human member of this family, ERO1-Lbeta. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lbeta gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 degrees C and to alleviate the "unfolded protein response" when expressed in ero1-1 cells indicates that ERO1-Lbeta is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lbeta display different tissue distributions. Furthermore, only ERO1-Lbeta transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.