A newly developed 30 S subunit reconstitution system using a complete set of recombinant proteins was used to study the ribosomal RNA (rRNA) neighborhood of ribosomal protein S5 in 30 S subunits and 70 S ribosomes by directed hydroxyl radical probing. Using three cysteine-containing mutant S5 proteins derivatized with 1-(p-bromoacetamidobenzyl)-Fe(II)-EDTA, we expanded on experiments carried out earlier using a natural protein reconstitution system. Natural 16 S rRNA, Fe(II)-S5, and the other recombinant ribosomal proteins were reconstituted into 30 S subunits. Both 30 S subunits and 70 S ribosomes containing Fe(II)-S5 were purified, and hydroxyl radicals were generated in situ from the tethered Fe(II). In 30 S subunits, 16 S rRNA nucleotides targeted by two positions on S5, C21 and C99, were virtually identical to those observed in the previous work, supporting the validity of the recombinant protein reconstitution system for probing studies. Interestingly, new cleavages were detected using Fe(II)-C129-S5, possibly reflecting incorporation of more derivatized protein into 30 S subunits due to the increased reconstitution efficiency of the recombinant protein system. These newly targeted positions overlap, but are distinct from, those observed using Fe(II) tethered to C21, which is near C129 in the S5 structure. In 70 S ribosomes, the cleavage pattern of 16 S rRNA was very similar to that observed in 30 S subunits for all target sites except for the absence of those at the extreme 5' end of 16 S rRNA. Additionally, probing of 70 S ribosomes from Fe-C99-S5 results in cleavage of 23 S rRNA in the 1690-1770 region of domain IV. These data provide constraints for the three-dimensional location of nucleotides within domain IV of 23 S ribosomal RNA relative to known features of the 30 S subunit.