To characterize ribosome-ligand interactions, we have used a cleavage reagent, 1,10-orthopenanthroline-Cu(II), tethered to various ligands, to cleave nearby regions of rRNA. The phenanthroline is tethered to the ligand using either an internal 4-thiouridine or a terminal thiophosphate. When Cu2+ and a reducing agent, such as mercaptopropionic acid, are present, cleavage of nearby nucleic acids occurs. The cleavage sites can be identified using primer-extension analysis. We have identified rRNA cleavage sites resulting from transcribed tRNAPhe having randomly placed phenanthroline-Cu(II), tRNAPhe with phenanthroline-Cu(II) at position 8, and a DNA oligomer complementary to positions 2655-2667 (alpha-sarcin region) with phenanthroline-Cu(II) placed at the 5' end. These results provide important new information on the structure of the rRNA within ribosomal subunits and on the proximity of rRNA neighborhoods to these bound ligands.