Studying the intricate folding of rRNA within the ribosome remains a complex problem. Phenanthroline-Cu(II) complexes cleave phosphodiester bonds in rRNA in specific regions, apparently especially where the rRNA structure is constrained in some fashion. We have introduced phenanthroline-copper complexes into 50S Escherichia coli ribosomal subunits and shown specific cleavages in the regions containing nucleotides 60-66 and 87-100. This specificity of cleavage is reduced when the ribosome is heated to 80 degrees C and reduced to background when the ribosomal proteins are extracted and the cleavage repeated on protein-free 23S rRNA. It has been suggested that nucleotides 60-66 and 87-95 in E.coli 23S rRNA are involved in a putative pseudoknot structure, which is supported by covariance data. The paired cleavages of nearly equal intensity of these two regions, when in the ribosome, may further support the existence of a pseudoknot structure in the 100 region of 23S rRNA.