Selenium which occurs in proteins as the amino acid, selenocysteine, is essential for numerous biological processes and for human health. A prominent 75Se-labeled protein detected in human T-cells migrated as a 15-kDa band by SDS-polyacrylamide gel electrophoresis. This protein subunit was purified and subjected to tryptic digestion and peptide sequence analyses. Sequences of tryptic peptides derived from the protein corresponded to a human placental gene sequence containing an open reading frame of 162 residues and a readthrough in-frame TGA codon. Three different peptide sequences of the 15-kDa protein corresponded to a nucleotide sequence located downstream of this codon, suggesting that the T-cell 15-kDa selenoprotein contains a selenocysteine residue encoded by TGA. Post-translational processing of the N-terminal portion of the predicted gene product to give the 15-kDa protein was suggested on the basis of molecular mass, amino acid analysis, and immunoblot assays of the purified protein. The 3'-untranslated region (UTR) of the gene encoding the 15-kDa protein contained a sequence that is very similar to the canonical selenocysteine-inserting sequence element. Computer analysis of transcript map data bases indicated that this gene was located on human chromosome 1. Its coding sequence showed no homology to known protein-encoding genes. The 15-kDa protein gene was expressed as mRNA in a wide range of tissues, with increased levels in the thyroid, parathyroid, and prostate-derived cells as evidenced by searches of partial cDNA sequences in public data bases. Genes corresponding to the 15-kDa selenocysteine-containing protein were found in mice and rats, while the corresponding genes in Caenorhabditis elegans and Brugia malayi contained a cysteine codon in place of TGA. The discovery of a new human selenoprotein provides an additional example of the role of selenium in mammalian systems.