We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis. These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA. Expression of C2252/C2253 23 S rRNA in E. coli severely compromises cell growth. Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes. Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes. To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E. coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA. As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically. Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes. These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA.