Mitochondria are present in the majority of eukaryotic organisms and dominates generation of cellular energy through the electron transport chain and oxidative phosphorylation. Mutations in nuclear and mitochondrial DNA encoding complex subunits or assembly factors may impair oxidative phosphorylation and cause energy depletion. Electrons can be transferred to the respiratory chain by NADH via complex I or through succinate and complex II. Succinate is not cell permeable, although by adding groups the molecule can obtain the property and ability to enter the mitochondrial matrix. Inherited isolated complex I deficiency is a heterogeneous and multisystemic disorder and progression may be rapid with fatal outcome. Various diseases originate in complex I deficiency, such as Leigh syndrome. Since treatment is limited and primarily symptomatic the possibility of increasing oxidative phosphorylation via succinate and complex II is relevant to investigate. The effect of cell permeable succinate prodrug NV357 on respiration and ATP production during inhibition of complex I were studied in vitro. Periphery blood mononuclear cells from blood donors were utilized as source of human mitochondria and complex I deficiency was simulated by rotenone inhibition. Energy production decreased by rotenone presence and the succinate prodrug NV357 elevated oxygen consumption and ATP quantity. Energy production were reduced subsequent antimycin A addition, thus indicating increasing effect upstream of complex III. Rotenone completely inhibits the complex I and will not represent the deficiency in patients. Investigation of the succinate prodrug in cells obtained from individuals suffering complex I deficiency ought to be determined prior in vivo trials. Individuals suffering mild and severe mutations affecting complex I could benefit from a cell permeable succinate prodrug. If this approach is successful and common diseases associated to mitochondrial dysfunction increase energy production via succinate and complex II the prodrug could benefit many.