We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2475-2483, a single-stranded sequence (the 2475 loop) near the peptidyltransferase center of Escherichia coli ribosomes. On photolysis in the presence of 50S subunits, it site-specifically incorporates into proteins L1, L13, L16, L32, and L33 and into 23S rRNA nucleotides G2470, A2471, and G2472. These results provide clear evidence that C2475 in 23S rRNA is within 21 A (the distance between C2475 and the photogenerated nitrene) of proteins L1, L13, L16, L32, and L33. The implications of these results for the evolving model of the internal structure of the 50S subunit are considered.