Activation of proton pumping by reconstituted and native membrane-bound Complex I was studied using optical electric potential- and pH-sensitive probes. We find that reconstituted Complex I has a delay in proton translocation, which is significantly longer than the delay in quinone reductase activity, indicating an initially decoupled state of Complex I. Studies of the amount of NADH required for the activation of pumping indicate the prerequisite of multiple turnovers. Proton pumping by Complex I was also activated by NADPH, excluding significant reduction of Complex I and a preexisting Δψ as activation factors. Co-reconstitution of Complex I and ATPase did not indicate an increased membrane permeability for protons in the uncoupled Complex I state. The delay in Complex I proton pumping activation was also observed in subbacterial vesicles. While it is negligible at room temperature, it strongly increases at a lower temperature. We conclude that Complex I undergoes a conversion from a decoupled state to a coupled state upon activation. The possible origins and importance of the observed phenomenon are discussed.