The sole member of the ankyrin (A) subfamily of transient receptor potential (TRP) channels, TRPA1, is Ca2+-permeable and present in the cerebral vascular endothelium.1, 2 TRPA1 is localized to sites of endothelial cell-smooth muscle communication called myoendothelial junctions.2 Upon activation with exogenous electrophilic compounds, TRPA1 evokes endothelium-dependent vasodilation,2 but endogenous agonists of the channel in this tissue remain elusive. TRPA1 channels have been shown to be activated by lipid peroxidation products (LPP)3, 4 formed from the oxidative degradation of membrane lipids by reactive oxygen species (ROS). Rat cerebral artery immunolabeling revealed the presence of NADPH oxidase isoform 2 (NOX2), a ROS-generating enzyme, in the endothelium within myoendothelial junctions (n = 3). In addition, a proximity ligation assay demonstrated localization of TRPA1 within 40 nm of NOX2, but not NOX4, in intact cerebral arteries (n = 5). We therefore tested the hypothesis that TRPA1 is activated by endogenous LPP generated from local production of ROS by NOX in cerebral arteries. To examine the effect of NOX activation on TRPA1 activity, unitary TRPA1 Ca2+ influx events (TRPA1 Ca2+ sparklets) were recorded from primary cerebral artery endothelial cells loaded with Ca2+ indicator Fluo-4 AM using total internal reflection fluorescence (TIRF) microscopy. TIRF recordings of cells were analyzed using custom software, LC_Pro, where TRPA1 Ca2+ sparklets were autodetected, and parameters such as whole-cell frequency (Ca2+ sparklets/cell/s) were calculated. Addition of NADPH, a substrate of NOX, to the extracellular solution increased TRPA1 Ca2+ sparklet frequency in endothelial cells (0.04 ± 0.02 Hz before vs. 0.28 ± 0.08 Hz after, n = 20-30). This increase in TRPA1 Ca2+ sparklet frequency was attenuated by pretreatment with the selective TRPA1 antagonist HC-030031 (HC) (0.03 ± 0.02 Hz, n = 14), indicating that activation of NOX stimulates TRPA1 Ca2+ influx in endothelial cells. We then examined the effect of NOX activation in isolated, pressurized (80 mmHg) rat cerebral arteries. Superfusion of NADPH in the bathing solution induced vasodilation in a concentration-dependent manner (EC50 = 1.2 µM). This response was diminished by pre-treatment with HC (61.3 ± 21.6% dilation (NADPH) vs. 8.7 ± 16.2% dilation (HC + NADPH), n = 3). The LPP 4-hydroxy-nonenal (4-HNE) also caused cerebral artery dilation (EC50 = 8.4 µM) that was abolished by pre-treatment with HC (51.4 ± 7.8% dilation (4-HNE) vs. 0.0 ± 11.6 % (HC + 4-HNE), n = 5). Together, our findings suggest that NOX-derived LPP generation dilates cerebral arteries through TRPA1 activation. All data are mean ± SE; values of n refer to the number of cells/vessels used per each experiment.