Methionine-R-sulfoxide reductases (MsrBs) catalyze a stereospecific reduction of methionine-R-sulfoxides to methionines in proteins. Mammals possess three MsrB genes. MsrB1 (SelR) is a selenoprotein located in the cytosol and nucleus, MsrB2 (CBS-1) is a mitochondrial protein, and MsrB3 is a recently identified protein with an unusual localization pattern. Human MsrB3 occurs in two protein forms, MsrB3A and MsrB3B, which can be targeted to the endoplasmic reticulum (ER) and mitochondria, respectively. These forms are generated by alternative first exon splicing that introduces contrasting N-terminal signal peptides. Herein, we characterized mouse MsrB3 and found no evidence of alternative splicing of its gene. The ER signal was located upstream of the predicted mitochondrial signal sequence in a single coding region, whose product was targeted to the ER. Although the mitochondrial signal could function if placed at the N-terminus, it did not target MsrB3 to mitochondria as part of the entire coding region. In addition, immunoblot assays detected no mitochondrial MsrB3 in examined mouse tissues. The data suggest that, in mice, MsrB3 is largely or exclusively an ER-resident protein, and that the reduction of methionine-R-sulfoxides in different cellular compartments is provided by individual MsrB isozymes. (C) 2004 Elsevier Inc. All rights reserved.