Selenium is an essential trace element with known antioxidant properties. Cytosolic thioredoxin reductase from mammalian cells is a dimeric flavin enzyme comprising a glutathione reductase-like equivalent elongated with 16 residues including the conserved carboxy-terminal sequence, Gly-Cys-SeCys-Gly, where SeCys is selenocysteine. Replacement of the SeCys residue by Cys in rat cytosolic thioredoxin reductase using site-directed mutagenesis and expression in Escherichia coli resulted in a functional mutant enzyme having about one percent activity with thioredoxin as a substrate through a major loss of Kcat and a shift in the pH optimum from 7 to 9. The truncated enzyme expected in selenium deficiency by the UGA mRNA codon for SeCys acting as a stop codon was also expressed. This enzyme lacking the carboxy-terminal SeCys-Gly dipeptide contained FAD but was inactive because the SeCys selenol is in the active site. These results show that selenium is essential for the activity of thioredoxin reductase, explaining why this trace element is required for cell proliferation by effects on thioredoxin-dependent control of the intracellular redox state, ribonucleotide reductase production of deoxyribonucleotides, or activation of transcription factors. The selenazol drug ebselen (2-phenyl-1,2 benzisoselenazol-3 (2H)-one) is a known glutathione (GSH) peroxidase mimic with antioxidant properties. The hydrogen peroxide reductase activity of human thioredoxin reductase was stimulated 15-fold by 2 microM ebselen. Glutaredoxins protect against oxidative stress by catalyzing reduction of protein mixed disulfides with GSH. The mechanism of glutaredoxins as efficient general GSH-mixed disulfide oxidoreductases may protect proteins from inactivation as well as play a major role in general redox signaling.