Three tRNA Ser's (tRNA Ser 1, tRNA Ser 3a and tRNA Ser 3b) were highly purified from Escherichia coli by the combined use of several column chromatographic systems, DEAE-Sephadex A-50, BD-cellulose and a reverse phase partition system. All three tRNA Ser's were charged with serine using aminoacyl-tRNA synthetases from baker's yeast and rat liver. tRNA Ser 1 which recognized codons of UCA and UCG, and less effectively UCU, contained uridin-5-oxyacetic acid as well as 2-methylthio- N 6-( Δ 2-isopentenyl)adenosine. On the other hand, both tRNA Ser 3a and tRNA Ser 3b recognized codons of AGU and AGC. tRNA Ser 3a contained N-[9-β- d - ribofuranosyl)purine-6- ylcarbamoyl]threonine , while tRNA Ser 3b contained a minor constituent similar to but different from this threonine-containing adenosine derivative. Chromatographic profiles of the complete digestion with ribonuclease T 1 suggested structural similarity between tRNA Ser 3a and tRNA Ser 3b.