tRNAPhe transcribed in a T7 RNA polymerase system has been modified in such a way that 4-thiouridines have randomly replaced unmodified uridines. These 4-thiouridines serve as sites for conjugation of the cleavage reagent 5-iodoacetamido-1,10-phenanthroline (IOP). 1,10-Phenantholine, when complexed with Cu2+ in a reducing environment, causes hydrolysis of nearby nucleic acids. We show here that tRNA-phenanthroline (tRNA-OP) conjugates, when bound in situ to the P- and E-sites of 70 S ribosomes, cause cleavage, mainly in domains I, III and V of 23 S ribosomal RNA (rRNA). The cleavage sites in domain V predominantly occur very close to or in the peptidyl-transferase region. The regions of domain I and III that are cleaved are apparently folded in the 50 S ribosomal subunit so as to be proximal to the peptidyl-transferase center. Most of the cleavage events occur whether the tRNA-OP conjugate is bound to ribosomes alone, or yeast tRNA is also present in the P/P hybrid state. Cleavages that occur only in the absence of yeast tRNA are limited to the 1100 region of domain II, and the 2800 region of domain VI. Cleavages that occur only in the presence of yeast occur in the 2170 region of domain V. The regions of 23 S rRNA in which tRNA-OP induced cleavage occur complement those sites shown by chemical protection and cross-liking to be in a close proximity to the tRNA. However, the cleavage approach allows a more versatile and expanded view of the near neighborhood of rRNA surrounding the tRNA. These results provide considerable information which will allow a more detailed modeling of the tertiary structure of the 50 S ribosomal subunit.