Previous experiments have shown that the phylogenetically conserved G2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of peptidyl-tRNA. In the studies presented here, site-directed mutations were introduced at two other conserved positions in 23 S rRNA, G2251 and U2585, that were previously implicated in interaction of the CCA acceptor end of tRNA with the 50 S subunit P site. The mutant 23 S rRNAs were characterized by determining (1) the in vivo phenotypes, (2) the ability of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro, using footprinting with allele-specific primer extension and (3) the ability of mutant ribosomes to catalyze peptide bond formation using a chimeric reconstitution approach. Mutations at either position confer a dominant lethal phenotype when the mutant 23 S rRNA is coexpressed with the endogenous wild-type 23 S rRNA. Mutations at 2585 disrupt binding of the wild-type (CCA) tRNA oligonucleotide fragment and cause a modest decrease in the peptidyl transferase activity of reconstituted ribosomes. By contrast, mutations at 2251 abolish both binding of the wild-type (CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment. In neither case was the loss of binding or peptidyl transferase activity suppressed by mutations in the tRNA oligonucleotide fragment. Chemical modification analysis revealed that mutations at 2251 perturb the reactivity of bases 2584 to 2586, providing further evidence that the 2250 loop of 23 S rRNA interacts, either directly or indirectly, with the 2585 region in the central loop of domain V of 23 S rRNA.