An enzyme preparation from Salmonella typhimurium catalyzes the conversion of 5-methylaminomethyl-2-thiouridine in tRNAs to 5-methylaminomethyl-2-selenouridine when supplemented with selenide and ATP. Similar preparations from a Salmonella mutant strain carrying a defective selD gene fail to catalyze this selenium substitution reaction. However, supplementation of the deficient enzyme preparation with the purified selD gene product (SELD protein) restored synthesis of seleno-tRNAs. In the absence of the complementary enzyme(s), the SELD protein catalyzes the synthesis of a labile selenium donor compound from selenide and ATP. 31P NMR studies show that among the products of this reaction are AMP and a compound containing selenium bonded to phosphorus. The reaction is completely dependent on the addition of both selenide and magnesium. The dependence of reaction velocity on ATP concentration shows sigmoidal kinetics, whereas dependence on selenide concentration obeys Michaelis-Menten kinetics indicating a Km value of 46 microM for selenide.