Our aim was to investigate the mechanism of the selenium incorporation into tRNA in vitro using partially purified enzyme preparations from Salmonella typhimurium. Chromatography of the cell extracts from these bacteria on phenyl Sepharose yielded active enzyme(s) which eluted at 0.2 M ammonium sulfate. Added E. coli or S. typhimurium bulk tRNA were used as substrates. Similar to the results obtained from the broken cell preparations, ATP and 0-acetyl-L-serine were required for full activity. 0-acetyl-L-serine can be converted to selenocysteine and the incorporation of selenium from radiolabeled selenite into tRNA was decreased by unlabeled L - selenocysteine suggesting that this amino acid may serve as a selenium donor utilized for selenation of tRNA. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that co-eluted with authentic 5-methylaminomethyl-2-selenouridine. The 5 - methylaminomethyl - 2 - selenouridine content of tRNA was higher in the case of broken cell preparations than in the case of the phenyl Sepharose column fraction of these preparations suggesting that probably an enzyme component required for full activity was partially lost upon the phenyl Sepharose column chromatography.