Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dose-dependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution.