Fungal gliotoxin (GT) is a potent inhibitor of the O(2)(-)-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47(phox), a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C betaII with p47(phox) on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O(2)(-)-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b(558) is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b(558) and cytosolic components (native cytosol or recombinant p67(phox), p47(phox), and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 microM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67(phox), p47(phox), and Rac2 with flavocytochrome b(558) is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b(558)'s ability to reconstitute O(2)(-) generation, supporting the conclusion at the cellular level. The flavocytochrome b(558) heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b(558), which is accessible only before oxidase activation.