Unfractionated Escherichia coli tRNA has been aminoacylated with lysine and preferentially acetylated at the epsilon-amino nitrogen of lysine by reaction with N-acetoxysuccinimide. After treatment with peptidyl-tRNA hydrolase, 90% of the aminoacylated tRNA molecules were Nepsilon-acetyl-Lys-tRNA. Post-ribosomal supernatant enzymes would not deacylate Nepsilon-acetyl-Lys-tRNA in the presence of AMP and PPi, even though such mixed enzymes could acylate, with lysine, tRNA which had been exposed to the acetylation reaction conditions. Poly(rA) stimulated the binding of Nepsilon-acetyl-Lys-tRNA to E. coli ribosomes. At the ribosome and tRNA concentrations used, Nepsilon-acetyl-Lys-tRNA was bound nearly as well as Lys-tRNA at 30 mM Mg2+; at 10 mM Mg2+, the analogue was bound one-half as well as Lys-tRNA. Both Lys-tRNA and Nepsilon-acetyl-Lys-tRNA reacted only slightly with puromycin at either 10 or 30 mM Mg2+. When Lys-tRNAE. coli or Nepsilon-acetyl-Lys-tRNAE. coli were added to rabbit reticulocyte cell-free protein synthesizing incubations, the incorporation of either amino acid into protein was complete within 5 min. The final incorporation level of the analogue was 82% that of the unmodified lysine. After protein synthesized in the presence of Nepsilon-acetyl-[14C]Lys-tRNA had been digested enzymatically to single amino acids, ion-exchange chromatography and paper electrophoresis showed that nearly all of the radioactivity was present as Nepsilon-acetyllysine. Gel filtration of the post-ribosomal supernatant revealed that most of the Nepsilon-acetyllysine radioactivity cochromatographed with tetrameric hemoglobin.