Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously. It is demonstrated that an E. coli 23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself.