In this work 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 2497-2505, a single-stranded sequence that has been shown to fall within the peptidyltransferase center of Escherichia coli ribosomes [Cooperman, B. S., Weitzmann, C. J., & Fernandez, C. L. (1990) in The Ribosome: Structure, Function, & Evolution (Hill, W. E., Dahlberg, A., Garrett, R. A., Moore, P. B., Schlesinger, D., & Warner, J. R., Eds.) pp 491-501, American Society of Microbiology, Washington]. On photolysis in the presence of 50S ribosomes, it site-specifically incorporates into protein L3 (identified by both SDS-PAGE and immunological methods) and into three separate 23S rRNA regions: specifically, nucleotides 2454; 2501, 2502, 2505, 2506; and 2583, 2584. These results provide clear evidence that G-2505 in 23S rRNA is within 24 A (the distance between G-2505 and the photogenerated nitrene) of protein L3 and of each of the nucleotides mentioned above and are of obvious importance in the construction of detailed three-dimensional models of ribosomal structure. The approach we present is general and can be applied to determining ribosomal components neighboring regions of rRNA that are susceptible to binding by complementary oligodeoxyribonucleotides, both in intact 30S and 50S subunits and in subunits at various stages of reconstitution.