We have used oligodeoxyribonucleotide probes to investigate possible interactions between chloramphenicol and portions of the rRNA contained within the peptidyltransferase center of the Escherichia coli ribosome. Oligodeoxyribonucleotide probes complementary to bases 2448-2454, 2468-2482, and 2497-2505 of 23 S rRNA were hybridized to 50 S subunits in situ. Probe binding was qualitatively assessed by sucrose gradient centrifugation. Each probe was shown to bind specifically with its intended binding site through digestion of the rRNA within the RNA/DNA hetero-duplexes with RNase H and analysis of the digestion fragments using gel electrophoresis. Competitive binding experiments were conducted between each probe and the antibiotics chloramphenicol and erythromycin. The binding of a probe complementary to bases 2497-2505 was attenuated by 70% upon the binding of chloramphenicol. A probe complementary to bases 2468-2482 showed an increase in binding of 14% while binding of a probe complementary to bases 2448-2454 was not affected by chloramphenicol binding. Erythromycin did not affect the binding of any of these probes to 50 S subunits. These results suggest that bases within the 2497-2505 region of 23 S rRNA in E. coli may be involved in a chloramphenicol/rRNA interaction.