The organization of the 5′ terminus region in the 16S rRNA was investigated using a series of RNA constructs in which the 5′ terminus was extended by 5 nt or was shortened to give RNA molecules that started at positions −5, +1, +5, +8, +14, or +21. The structural and functional effects of the 5′ extension/truncations were determined after the RNAs were reconstituted. 30S subunits containing 16S rRNA with 5′ termini at −5, +1, +5, +8 and +14 had similar structures (judged by UV-induced crosslinking) and exhibited a gradual reduction in tRNA binding activity compared to that seen with 30S subunits reconstituted with native 16S rRNA. To create the 5′ terminal site-specific photocrosslinking agent, the reagent azidophenacylbromide (APAB) was attached to the 5′ terminus of 16S rRNA through a guanosine monophosphorothioate and the APA-16S rRNAs were reconstituted. Crosslinking carried out with the APA revealed sites in six regions around positions 300–340, 560, 900, 1080, the 16S rRNA decoding region, and at 1330. Differences in the pattern and efficiency of crosslinking for the different constructs allow distance estimates for the crosslinked sites from nucleotide G9. These measurements provide constraints for the arrangement of the RNA elements in the 30S subunit. Similar experiments carried out in the 70S ribosome resulted in a five- to tenfold lower frequency of crosslinking. This is most likely due to a repositioning of the 5′ terminus upon subunit association.