Glutathione peroxidase (EC 1.11.1.9) is one of a unique group of prokaryotic and eukaryotic enzymes that contain the unusual amino acid selenocysteine. The genes for these selenoproteins encode for the atypical amino acid at a TGA codon (UGA in the mRNA transcripts), which normally functions as a termination signal. The present studies analyzed the functional importance of sequences in the coding and 3'-untranslated regions of transcripts of the primary human cellular glutathione peroxidase gene (GPX1) to the insertion of selenocysteine at this UGA codon. Deletions in potential stem-loop or hairpin structures in the coding region did not substantially diminish incorporation of selenocysteine into glutathione peroxidase transiently expressed by the pCMV4 vector in COS-1 cells. However, selenocysteine insertion was completely abolished by deletion of four-nucleotide sequences in the 3'-untranslated region from within a conserved "selenocysteine insertion sequence" motif also found in the 3'-untranslated region of mammalian genes for other selenoproteins. Moreover, in constructs fusing the glutathione peroxidase 3'-untranslated region to the coding region of rab5b (an unrelated protein normally without any selenium moiety), the glutathione peroxidase 3'-untranslated region was sufficient to direct the translation of an opal (UGA) mutation as selenocysteine. Thus, our data directly demonstrate the importance of the selenocysteine insertion motif in the glutathione peroxidase gene and specifically show that sequence elements in the 3'-untranslated region are both necessary and sufficient for translational insertion of selenocysteine at a UGA codon in eukaryotic mRNA.