The effects of Mg2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis. Mg2+ concentrations between 1 and 20 mM and temperatures between 5 and 55 degreesC had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes. In contrast, two cross-links, C967 x C1400 and C1402 x C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg2+, increase in frequency at higher Mg2+ concentration, and are both more frequent when 50S subunits are included in the reactions. In 70S ribosomes, the cross-link C1402 x C1501 increases but the cross-link C967 x C1400 decreases at higher Mg2+ concentrations. One cross-link, C1397 x U1495, is detected only in 70S ribosomes and decreases in frequency as Mg2+ concentration is increased. An additional cross-link, A1093 x C1182, decreases upon subunit association. The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg2+ ions and subunit association.