To investigate the energy-conserving function of the NADH:ubiquinone reductase (complex I), we have selected oxonol VI [bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol] as the most sensitive probe for measuring the reactions of membrane potential generation in submitochondrial particles. Calibration of the oxonol signals with potassium diffusion potentials shows a non-linear response after a threshold around -50 mV. Thermodynamic evaluations indicate that the upper limit of the oxonol response to the potential generated by complex I is around -220 mV, which is close to the maximal protonmotive force in coupled submitochondrial particles. NADH addition to particles in which ubiquinol oxidation is blocked by inhibitors of other respiratory complexes generates oxonol signals corresponding to membrane potentials of -130 to -180 mV. These signals are produced by about four turnovers of the complex reducing endogenous ubiquinone (i.e. non-steady-state conditions) and are equivalent to a charge separation similar to that of the antimycin-sensitive reactions of ubiquinol:cytochrome c reductase (complex III). The transient oxonol signals under non-steady-state conditions are thus informative of crucial steps in the electrogenic reactions catalyzed by complex I. The possible nature of these electrogenic reactions is discussed in relation to proposed mechanisms for complex I.