Recent atomic models of ribosomal structure emphasize the need for new biochemical methods, suitable for fine-scale studies of ribosomal structure and function. We have used the phosphorothioate approach to probe iodine accessibility of 23 S rRNA domain I phosphates inside functional 50 S ribosomal subunits. Five percent of R(P) isomers of nucleoside phosphorothioate were incorporated into Thermus aquaticus 23 S rRNA during in vitro transcription. Ribosomal large subunits were reconstituted from 23 S rRNA and 5 S rRNA transcripts and ribosomal large subunit proteins. The resulting particles sedimented as 50 S and were active in a peptide bond formation assay. Iodine-induced cleavage sites were determined for domain I of 23 S rRNA by reverse transcriptase-directed primer extension. Specific signals were detected at 360 positions, 80 of which were protected in reconstituted 50 S subunits. We argue that most observed protections are caused by shielding of phosphates by ribosomal proteins. The phosphorothioate approach can be extended to analyze dynamic structural changes during translation and the functional roles of individual chemical groups in rRNA.