The rates of adenosine triphosphate (ATP) production by isolated mitochondria and mitochondrial creatime kinase incubated in isotopically pure media containing, separately, 24Mg2+, 25Mg2+, and 26Mg2+ ions were shown to be strongly dependent on the magnesium nuclear spin and magnetic moment. The rate of adenosine 5′-diphosphate phosphorylation in mitochondria with magnetic nuclei25Mg is about twice higher than that with the spinless, nonmagnetic nuclei24.26Mg. When mitochondrial oxidative phosphorylation was selectively blocked by treatment with 1-methylnicotine amide, 25Mg2+ ions were shown to be nearly four times more active in mitochondrial ATP synthesis than 24,26Mg2+ ions. The rate of ATP production associated with creatine kinase is twice higher for 25Mg2+ than for 24.26Mg and does not depend on the blockade of oxidative phosphorylation. There is no difference between 24Mg2+ and 26Mg2+ effects in both oxidative and substrate phophorylation. These observations demonstrate that the enzymatic phosphorylation is a nuclear spin selective process controlled by magnetic isotope effect. The reaction mechanism proposed includes a participation of intermediate ion-radical pairs with Mg+ cation as a radical partner. Therefore, the key mitochondrial phosphotransferases work as a magnesium nuclear spin mediated molecular machines.