Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNA(Sec) to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec-tRNA(Sec) in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS-dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes.