The membrane potential (ΔΨm) 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 H₂O₂ 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 H₂O₂ formation from 207 ± 4.5 to 312 ± 12 pmol/min/mg protein. Depolarization by FCCP (5–250 nM) in the presence of BSA decreased H₂O₂ formation but only to the level observed in the absence of BSA. Rotenone stimulated the formation of H₂O₂ both in the absence and presence of BSA. It is suggested that H₂O₂ formation in mitochondria supported by NADH-linked substrates is sensitive to changes in ΔΨm only when mitochondria are highly polarized and even then, 60% of ROS generation is independent of ΔΨm. This is in contrast to earlier reports on the highly ΔΨm sensitive ROS formation related to reverse electron flow observed in well-coupled succinate-supported mitochondria.