A range of antibiotic inhibitors that act within the peptidyl transferase center of the ribosome were examined for their capacity to perturb the relative positioning of the 3′ end of P/P′-site-bound tRNA and the Escherichia coli ribosome. The 3′-terminal adenosines of deacylated tRNA and N-Ac-Phe-tRNA were derivatized at the 2 position with an azido group and the tRNAs were cross-linked to the ribosome on irradiation with ultraviolet light at 365 nm. The cross-links were localized on the rRNA within extended versions of three previously characterized 23S rRNA fragments F1′, F2′, and F4′ at nucleotides C2601/A2602, U2584/U2585 (F1′), U2506 (F2′), and A2062/C2063 (F4′). Each of these nucleotides lies within the peptidyl transferase loop region of the 23S rRNA. Cross-links were also formed with ribosomal proteins L27 (strong) and L33 (weak), as shown earlier. The antibiotics sparsomycin, chloramphenicol, the streptogramins pristinamycin IA and IIA, gougerotin, lincomycin, and spiramycin were tested for their capacity to alter the identities or yields of each of the cross-links. Although no new cross-links were detected, each of the drugs produced major changes in cross-linking yields, mainly decreases, at one or more rRNA sites but, with the exception of chloramphenicol, did not affect cross-linking to the ribosomal proteins. Moreover, the effects were closely similar for both deacylated and N-Ac-Phe-tRNAs, indicating that the drugs selectively perturb the 3′ terminus of the tRNA. The strongest decreases in the rRNA cross-links were observed with pristinamycin IIA and chloramphenicol, which correlates with their both producing complex chemical footprints on 23S rRNA within E. coli ribosomes. Furthermore, gougerotin and pristinamycin IA strongly increased the yields of fragments F2′ (U2506) and F4′ (U2062/C2063), respectively. The results obtained with an RNAse H approach correlate well with primer extension data implying that cross-linking occurs primarily to the bases. Both sets of data are also consistent with the results of earlier rRNA footprinting experiments on antibiotic–ribosome complexes. It is concluded that the antibiotics perturb the relative positioning of the 3′ end of the P/P′-site-bound tRNA and the peptidyl transferase loop region of 23S rRNA.