The substrate specificity towards RNA/DNA chimeric oligonucleotide containing stereodefined phosphorothioate bond of the RP and SP configuration at the scissile site was determined for the cleavage reaction catalyzed by the deoxyribozyme 10-23. Single-turnover kinetics in the presence of either Mg2+or Mn2+ was used to determine the thio- and manganese rescue effects of the cleavage reaction. The obtained results indicate that the unmodified substrate and its SP thio-analog are specifically and efficiently cleaved even at low metal ion concentration (0.02 mM). By contrast, the presence of the phosphorothioate bond of the RP configuration at the scissile site caused complete resistance of this substrate in the deoxyribozyme-mediated cleavage reaction in a broad metal ion concentration (from 0.02 to 100 mM). These findings indicate the lack of a direct metal ion coordination to the non-bridging phosphate oxygens of the scissile phosphate bond (no β catalysis is observed). Plausible mechanism of this 10-23 deoxyribozyme-assisted catalysis reaction involves engagement of the divalent metal cation in the coordination of the oxygen of the 2'-OH group and migration of proton to the pro-RP oxygen of the scissile phosphate.