The phenylalanine tRNA of Rhodopseudomonas sphaeroides was fractionated on benzoylated diethylaminoethyl-cellulose into four isoaccepting species (tRNAPheI to IV). tRNAPheIII represented 80% of the total tRNAPhe in anaerobic, photosynthetically grown organisms, whereas in cultures grown aerobically for prolonged periods, tRNAPheII represented 80% of the total. In cultures adapting to aerobic growth, the addition of rifampin resulted in a tRNAPhe profile characteristic of anaerobic-photosynthetic conditions due to the conversion of tRNAPheII to tRNAPheIII. In fully adapted aerobic cultures, this conversion was inhibited in the presence of chloramphenicol or rifampin. The conversion of tRNAPheIII to tRNAPheII was not observable in vivo. It is proposed that an enzymic activity synthesized during anaerobic-photosynthetic growth was responsible for the conversion.