At high concentrations, Mg²⁺ ions are known to stabilize the structure of the 16S rRNA in a con- formation favorable for binding to the ribosomal proteins in the course of the eubacterial 30S ribosomal subunits assembly in vitro. The effect of Mg²⁺ on the formation of the 18S rRNA structure during 40S subunit assembly remains poorly explored. In this work, we show that a sequential increase in the Mg²⁺ concentration from 0.5 to 20 mM leads to a significant decrease in the affinity of recombinant human ribosomal protein S13 (rpS13e) to the RNA transcript that corresponds to the central domain fragment of 18S rRNA (18SCD). The regions near the rpS13e binding site in 18SCD, including the nucleotides of helices H20 and H22, the availability of which to hydroxyl radicals depended on the Mg²⁺ concentration, were determined. The increase in the Mg²⁺ concentration was shown to result in the enhanced accessibility of nucleotides G933-C937 and C1006-A1009 in helix H22 and reduced accessibility of nucleotides A1023, A1024, and C1026-A1028 in the H20 helix. A comparison of the obtained results with crystallographic data of the central domain of 18S rRNA in the 40S ribosomal subunit of Saccharomyces cerevisiae has led to the conclusion that an increase in the Mg²⁺ concentration resulted in the reorientation of helices H20 and H24 relative to to helices H22 and H23 and the formation of a structure in which these helices were positioned the same way as in 40S subunit. Hence, the saturation of the central domain of 18S rRNA with coordinated Mg²⁺ ions causes the same changes in its structure as rpS13e binding, and results in the decreased affinity of this domain to the protein.