The effect of correlations between kinetic parameters of two inducible substrates on allosteric activation of EcoRII endonuclease hydrolysis of one substrate was studied. The pairs of DNA duplexes were constructed that were the substrates of EcoRII restriction endonuclease or their analogs and had different kinetic constants of interaction with the enzyme; the effects of their concentrations on mutual hydrolysis induction were studied. A kinetic mechanism is suggested considering the allosteric effects of two DNA recognition sites on dimeric molecule of EcoRII. Mathematic modeling was used to analyze the kinetic mechanism and evaluate optimal characteristics of the inductor. Thus, activation increases when (i) substrate concentration decreases, (ii) enzyme binding of two inductor or substrate molecules decreases, (iii) binding of one substrate molecule increases versus binding of one inductor molecule, and (iv) kcat of the enzyme-substrate complex including on substrate and one inductor increases.