Peptidyl transferase, the ribosomal activity responsible for catalysis of peptide bond formation, is resistant to vigorous procedures that are conventionally employed to remove proteins from protein-nucleic acid complexes. When the "fragment reaction" was used as a model assay for peptide bond formation, Escherichia coli ribosomes or 50 S subunits retained 20 to 40 percent activity after extensive treatment with proteinase K and SDS, but lost activity after extraction with phenol or exposure to EDTA. Ribosomes from the thermophilic eubacterium Thermus aquaticus remained more than 80 percent active after treatment with proteinase K and SDS, which was followed by vigorous extraction with phenol. This activity is attributable to peptidyl transferase, as judged by specific inhibition by the peptidyl transferase-specific antibiotics chloramphenicol and carbomycin. In contrast, activity is abolished by treatment with ribonuclease T1. These findings support the possibility that 23 S ribosomal RNA participates in the peptidyl transferase function.