75Se-Labeled tRNAs were synthesized by Clostridium sticklandii cultures supplemented with 1 microM sodium [75Se]selenite or [75Se]selenocysteine. This process is highly specific for selenium; it occurred in the presence of 1.2 mM sodium sulfide and was not decreased by the further addition of a 500-fold molar excess of cysteine. The 75Se in these tRNAs was located in the polynucleotide portion of the molecules and not in esterified (alkali-labile) selenocysteine. Inhibition of cell multiplication by antibiotics that block either protein synthesis or DNA-dependent RNA synthesis did not prevent this 75Se incorporation. Three [75Se]tRNAs were separated from C. sticklandii cells labeled in the presence of chloramphenicol and were partially purified by chromatography on benzoylated DEAE-cellulose and DEAE-Sephadex A-50 columns. These were designated seleno-tRNAs I, II, and III according to their elution sequence from benzoylated DEAE-cellulose. Cochromatography of purified seleno-tRNA II on DEAE-Sephadex A-50 with an L-proline-accepting species suggests that it is a selenium-containing L-prolyl-tRNA.