Unmodified uridines have been randomly replaced by 4-thiouridines in transfer RNAPhe (tRNAPhe) transcribed in a T7 RNA polymerase system. These 4-thiouridines serve as conjugation sites for attachment of the cleavage reagent 5-iodoacetamido-1,10-o-phenanthroline (IoP). In a reducing environment, when complexed with Cu2+, 1,10-o-phenanthroline causes cleavage of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-oP) conjugates, when bound at the P-site of 70S ribosomes and 30S ribosomal subunits, caused cleavage of ribosomal RNA (rRNA) mainly in domains I and II of 16S rRNA. Some positions were cleaved only when tRNA-oP was bound to 70S ribosomes or to 30S ribosomal subunits. In domain I, most cleavage sites occurred in or near the 530 pseudoknot region. In domain II, most nucleotides cleaved were near the 690 region and the 790 region. The only positions cleaved in domain III were near the 1050 region. There were no discernible nucleotides cleaved near the 1400 (decoding) region. Our results corroborated results of others, which have shown these sites to be protected from chemical modification by tRNA binding or to be cross-linked to P-site-bound tRNA. Use of cleavage reagents tethered to tRNA provides evidence for additional regions of rRNA that may be proximal to bound tRNA.