It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na(+) uptake, indicating that a redox-driven, primary Na(+) pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 mumol min(-1) mg(-1)) to Na(+) translocation (2.0 mumol min(-1) mg(-1)). NADH-driven Na(+) transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na(+) pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na(+) gradient established by complex I.