(S)-alpha-Hydrazinophenylalanyl-tRNA(Phe), an amino acyl-tRNA derivative containing the unnatural amino acid (S)-alpha-hydrazinophenylalanine, was prepared in an effort to examine the stereochemical requirements of the A-site of the ribosome during in vitro protein synthesis. The (S)-alpha-hydrazinophenylalanine moiety was of interest because it contains two nucleophilic centers, the secondary nitrogen attached to Ca, which is normally acylated during the course of peptide bond formation, and the sterically less hindered primary nitrogen. To determine the position of acylation, (S)-alpha-hydrazinophenylalanyl-tRNA(Phe) was tested in an Escherichia coli in vitro protein biosynthesizing system lacking elongation factor G, such that only dipeptide products were formed. The dipeptide product mixture was analyzed by HPLC in direct comparison with authentic synthetic standards. The dipeptide assay utilizing (S)-alpha-hydrazinophenylalanyl-tRNA(Phe) as the A-site tRNA established that the analogue functioned well as an acceptor tRNA; HPLC analysis of the products showed that both dipeptides were formed in approximately equal amounts. When attached to a suppressor tRNA transcript, (S)-alpha-hydrazinophenylalanine was also incorporated into position 27 of dihydrofolate reductase in an E. coli protein synthesizing system by readthrough of a nonsense codon. This finding expands the currently accepted model of peptide bond formation at the ribosome and adds to the repertoire of peptide-like products shown to form at the peptidyltransferase center of the ribosome.