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Herein we present the results of studying the effects of different isotopes of magnesium, magnetic 25Mg and nonmagnetic 24Mg and 26Mg, at concentrations of Mg2+ up to 10 mM, on bioluminescence spectra and enzymatic activity of firefly luciferase. Neither kinetics of the bioluminescent signal nor the bioluminescence spectra in the luciferase-catalyzed oxidation of luciferin have manifested any statistically significant dependence on the type of magnesium isotope, magnetic 25Mg or nonmagnetic 24Mg or 26Mg. Further still, no MIEs have been revealed in the luciferase-catalyzed oxidation of luciferin. Thus, it means that firefly luciferase bioluminescence can serve as the feasible and adequate tool for search and studies of magnetic isotope effects in ATP-dependent enzyme reactions in biological systems, including the enzymatic synthesis and hydrolysis of ATP.