From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low-K+(5 mm KCl) medium there was a large decay of NADH and a 2.5-fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate-dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH-dependent *O2- production of CGC lysates, increased 2.5- to threefold. The stimulation of the ascorbate-dependent NADH oxidase activity by oxidized cytochrome c, 2.5-fold at saturation with a K(0.5) of 4-5 microm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate-dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and *O2- release) of CGC lysates. 4-Hydroxyquinazoline (4-HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4-HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4-HQ of the ascorbate-dependent NADH oxidase activity of CGC lysates, showing the same K(0.5)-value (4-5 mm 4-HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of *O2- overproduction by the plasma membrane ascorbate-dependent NADH oxidase.