Complex I (NADH-ubiquinone oxidoreductase) in the respiratory chain of mitochondria and several bacteria functions as a redox-driven proton pump that contributes to the generation of the protonmotive force across the inner mitochondrial or bacterial membrane and thus to the aerobic synthesis of ATP. The stoichiometry of proton translocation is thought to be 4 H(+) per NADH oxidized (2 e(-)). Here we show that a H(+)/2 e(-) ratio of 3 appears more likely on the basis of the recently determined H(+)/ATP ratio of the mitochondrial F(1)F(o)-ATP synthase of animal mitochondria and of a set of carefully determined ATP/2 e(-) ratios for different segments of the mitochondrial respiratory chain. This lower H(+)/2 e(-) ratio of 3 is independently supported by thermodynamic analyses of experiments with both mitochondria and submitochondrial particles. A reduced H(+)/2 e(-) stoichiometry of 3 has important mechanistic implications for this proton pump. In a rough mechanistic model, we suggest a concerted proton translocation mechanism in the three homologous and tightly packed antiporter-like subunits L, M, and N of the proton-translocating membrane domain of complex I.