Reactive oxygen species (ROS) play a central role in the genesis of vascular diseases such as hypertension, atherosclerosis, and restenosis after angioplasty. The main enzymatic source of ROS in vascular smooth muscle cells (VSMC) is the multisubunit complex NAD(P)H oxidase, whose activity is increased by growth factors and hypertrophic stimuli such as angiotensin II (Ang-II). Understanding molecular and cellular mechanisms of NAD(P)H oxidase control is thus essential to clarify the pathophysiology of vascular redox processes. We recently described close functional/spatial association between NAD(P)H oxidase and protein disulfide isomerase (PDI), a dithiol-disulfide oxidoreductase chaperone of the endoplasmic reticulum (ER) and provided evidence that such interaction couples oxidative stress to ER stress. However, the cellular processes governing PDI/NAD(P)H oxidase interaction remain unclear. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth, differentiation and apoptosis. Inhibition of proteasome activity has been associated with apoptosis in various cell types. Here, we studied the effect of proteasome inhibitors in NADPH oxidase activity, expression of PDI and ER-resident KDEL proteins and cell viability. HPLC analysis of dihydroethidium (DHE) oxidation products (a superoxide assay) in intact VSMC revealed that lactacystin and MG132 increased DHE oxidation rates after 4hs (ca. 70% and 46% vs control, respectively). Newly-validated DHE assays showed that VSMC incubation with lactacystin resulted in increased VSMC membrane NADPH oxidase activity (ca. 22% vs control). Western-blotting analysis of total VSMC cell homogenate confirmed that the proteasome inhibitors lactacystin (5m M) and MG132 (10m M) induced overexpression of KDEL proteins after 16hs. Finally, MTT Cell Proliferation Assay showed decrease of the cell viability after incubation with lactacystin and MG132 for 48hs (ca. 46% and 70% vs control, respectively). In conclusion, proteasome inhibition leads to early increase in NADPH oxidase activity and superoxide production, associated to induction of ER chaperones and subsequent cell death in VSMC. Together, these results suggest for the first time that ubiquitin-proteasome system may be relevant to modulate PDI/NAD(P)H oxidase interaction in the context of ER stress (Supported by FAPESP, CNPq Milênio Redoxoma).