Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5 -guanosine-alpha-phosphorothioate at the junction between the two fragments, and reconstituted 30S subunits with the two separate pieces of RNA and the small subunit proteins. Reconstituted 30S subunits capable of association with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved sites in the 16S rRNA backbone that were close in three-dimensional space to the Fe(II), and a primer extension was used to identify these sites of cleavage. Two sets of 16S ribosomal RNA fragments, 1-360/361-1542 and 1-448/449-1542, were reconstituted into active 30S subunits. Fe(II) tethered to position 361 results in cleavage of 16S rRNA around nucleotides 34, 160, 497, 512, 520, 537, 552, and 615, as well as around positions 1410, 1422, 1480, and 1490. Fe(II) tethered to position 449 induces cleavage around nucleotide 488 and around positions 42 and 617. Fe(II) tethered to the 5 end of 16S rRNA induces cleavage of the rRNA around nucleotides 5, 601, 615, and 642. These results provide constraints for the positioning of these regions of 16S rRNA, for which there has previously been only limited structural information, within the 30S subunit.