The phagocytic NADPH oxidase generates superoxide by transferring electrons from cytosolic NADPH to extracellular O2. The activity of the oxidase at the plasma membrane can be measured as electron current (Ie), and the voltage dependence of Ie was recently reported to exhibit a strong rectification in human eosinophils, with the currents being nearly voltage independent at negative potentials. To investigate the underlying mechanism, we performed voltage-clamp experiments on inside-out patches from human eosinophils activated with PMA. Electron current was evoked by bath application of different concentrations of NADPH, whereas slow voltage ramps (0.8 mV/ms), ranging from −120 to 200 mV, were applied to obtain ‘steady-state’ current–voltage relationships (I–V). The amplitude of Ie recorded at −40 mV was minimal at 8 μM NADPH and saturated above 1 mM, with half-maximal activity (Km) observed at approx. 110 μM NADPH. Comparison of I–V values obtained at different NADPH concentrations revealed that the voltage-dependence of Ie is strongly influenced by the substrate concentration. Above 0.1 mM NADPH, Ie was markedly voltage-dependent and steeply decreased with depolarization within the physiological membrane potential range (−60 to 60 mV), the I–V curve strongly rectifying only below −100 mV. At lower NADPH concentrations the I–V curve was progressively shifted to more positive potentials and Ie became voltage-independent also within the physiological range. Consequently, the Km of the oxidase decreased by approx. 40% (from 100 to 60 μM) when the membrane potential increased from −60 to 60 mV. We concluded that the oxidase activity depends on both membrane potential and [NADPH], and that the shape of the Ie–V curve is influenced by the concentration of NADPH in the submillimolar range. The surprising voltage-independence of Ie reported in whole-cell perforated patch recordings was most likely due to substrate limitation and is not an intrinsic property of the oxidase.