Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31]S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.