The membrane potential (DeltaPsim) dependence of the generation of reactive oxygen species (ROS) in isolated guinea-pig brain mitochondria respiring on NADH-linked substrates (glutamate plus malate) was addressed. Depolarization by FCCP was without effect on H(2)O(2) formation in the absence of bovine serum albumin (BSA). Addition of BSA (0.025%) to the assay medium hyperpolarized mitochondria by 6.1 +/- 0.9 mV (from 169 +/- 3 to 175.1 +/- 2.1 mV) and increased the rate of H(2)O(2) formation from 207 +/- 4.5 to 312 +/- 12 pmol/min/mg protein. Depolarization by FCCP (5-250 nM) in the presence of BSA decreased H(2)O(2) formation but only to the level observed in the absence of BSA. Rotenone stimulated the formation of H(2)O(2) both in the absence and presence of BSA. It is suggested that H(2)O(2) formation in mitochondria supported by NADH-linked substrates is sensitive to changes in DeltaPsim only when mitochondria are highly polarized and even then, 60% of ROS generation is independent of DeltaPsim. This is in contrast to earlier reports on the highly DeltaPsim sensitive ROS formation related to reverse electron flow observed in well-coupled succinate-supported mitochondria.