tRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution-interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA(Phe) from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA(Phe) and E. coli tRNA(fMet) indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA(Phe), we propose that specific Rp-phosphate oxygens are required for anticodon loop ("U-turn") stabilization or are involved in interactions with the ribosome on correct tRNA-mRNA complex formation.