Broken cell preparations of Salmonella typhimurium rapidly incorporated radiolabeled selenium from radiolabeled selenite into tRNA by an ATP-dependent process. Selenium incorporation in the presence of radiolabeled selenite was enhanced by the selenocysteine precursor, O-acetyl-L-serine. This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of radiolabeled selenium from radiolabeled selenite was decreased by the addition of L-selenocysteine but not by the D isomer. When homologous bulk tRNA was added to the broken cell preparations, an increased rate of radiolabeled selenium labeling was observed. The supernatant fraction of the broken cell preparation contained all of the enzymes required for this process. 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.