NAD(P)H oxidase is a multicomponent enzyme which catalyses the generation of superoxide in phagocytes. Upon activation, a cytosolic complex consisting of the oxidase subunits p47^phox, p67^phox and p40^phox together with Rac2 associates with the membrane-bound cytochrome b588 comprised of p22^phox and gp91^phox. NAD(P)H oxidase activation is inhibitable by thiol oxidants, not affected by the global redox cell state. Thiol oxidoreductases are effective modulators of the thiol redox state on cell membranes, endoplasmic reticulum (ER) and cytosol. Since spontaneous thiol-disulfide exchange reactions tend to be sufficiently slow and nonspecific to account for efficient target protein regulation, we hypothesized that thiol oxidoreductases could regulate NAD(P)H oxidase activity. The goal of the present study was to investigate the role of protein disulfide isomerase (PDI), a ubiquitous multifunctional protein of the thioredoxin family, in the activation of neutrophil NAD(P)H oxidase Western Blotting confirmed robust PDI expression in neutrophils. PDI antagonism with bacitracin or scrambled RNase led to 60-100 % inhibition (p<0.05) of NAD(P)H oxidase activity in a recombinant cell free system assessed with SOD inhibitable cytochrome c reduction. Neutrophil activation with phorbol myristate acetate (PMA) led to a significant increase in oxidase activity, which was followed by the translocation of PDI, p47^phox and p67^phox, from the cytosol to the membrane. Co-immunoprecipitation experiments using anti-PDI and anti-oxidase subunit antibodies indicated close spatial interaction between PDI and the oxidase subunits p67^phox and p47^phox. PDI is normally an ER protein that can migrate to plasma membrane. Immunofluorescence labeling showed that in neutrophils PDI is co-localized with the p22^phox oxidase subunit. This co-localization is increased in PMA stimulated neutrophils. ER stress is a situation where PDI expression and/or membrane traffic is increased. Incubation of neutrophils with ER stressors thapsigargin (5mM) or tunicamycin (5mg/ml) led to marked time-dependent (0-120 min) increase in NAD(P)H oxidase activity (p< 0.05). Therefore, PDI displays spatial and functional interaction with the oxidase. In particular, PDI can modulate neutrophil NAD(P)H oxidase activation. Altogether these results suggest that PDI closely associates with neutrophil NAD(P)H oxidase and could act as a novel redox sensitive regulatory protein potentially affecting subunit translocation and superoxide generation. Financial Support: CNPq, FAPESP.