A photo‐reactive diazirine derivative was attached to the 2‐thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli. This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes. After photo‐activation of the diazirine label, the sites of cross‐linking to 16S rRNA were identified by our standard procedures. Each of the three tRNA binding sites showed a characteristic pattern of cross‐linking. From tRNA at the A site, a major cross‐link was observed to position 1378 of the 16S RNA, and a minor one to position 936. From the P site, there were major cross‐links to positions 693 and to 957 and/or 966, as well as a minor cross‐link to position 1338. The E site bound tRNA showed major cross‐links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross‐link observed from the A site). Immunological analysis of the concomitantly cross‐linked ribosomal proteins indicated that S7 was the major target of cross‐linking from all three tRNA sites, with S11 as a minor product. The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.