A method for binding tRNA to ribosomes, introduced by Watanabe [Watanabe, S. (1972) J. Mol. Biol. 67, 443-457], permits nonenzymatic binding of N-acetyl-Phe-tRNA(Phe) to either the ribosomal aminoacyl-tRNA (A) or peptidyl-tRNA (P) site with almost 100% specificity. We used this method to analyze a possible codon-anticodon interaction at the P site for NH(2)-blocked aminoacyl-tRNA and deacylated tRNA. N-Acetyl-Phe-tRNA(Phe) bound only to the P site of poly(U)-programmed 70S ribosomes, not to poly(A)-programmed ribosomes. The reverse mRNA dependence was found for N-acetyl-Lys-tRNA(Lys). A series of purified deacylated tRNAs was analyzed in the poly(U) and poly(A) system for abilities to block P-site binding of N-acetyl-aminoacyl-tRNA and to direct the N-acetyl-aminoacyl-tRNA to the A site. Only the cognate tRNA was as effective as the bulk tRNA at a concentration of less than 1/20th that of bulk tRNA. tRNAs whose corresponding codons are identical or similar (same base character) in the first two codon positions showed a low but significant effect. The other noncognate tRNAs were unable to direct the NH(2)-blocked aminoacyl-tRNAs to the A site. Chlortetracycline interfered neither with the P-site binding of NH(2)-blocked aminoacyl-tRNA nor with the effects of deacylated tRNAs. Furthermore, the translocation blocker viomycin affected neither the binding to the A site nor that to the P site. These effects of both antibiotics indicate that both kinds of tRNA do not bind transiently in the A site before filling the P site and that codon-anticodon interaction takes place at the P site.