The phosphoramidate of CCdAp and puromycin (CCdApPuro) is a potent inhibitor of ribosomal peptidyl transferase, as assayed by the fragment reaction. Inhibition is competitive at the ribosomal A-site. CCdApPuro protects P-site-associated bases in the peptidyl transferase loop region of 23S rRNA from carbodiimide modification. The Ki's of structural homologues of CCdApPuro suggest that both the CCdA and puromycin moieties participate in binding. Thus, CCdApPuro appears to bridge the A- and P-sites of the ribosome, implying that substrates are juxtaposed with a geometry suitable for direct reaction during peptidyl transfer.