The mammalian transient receptor potential (TRP) superfamily of cation channels consists of 28 members divided among six subfamilies based on sequence homology. The function of many TRP channels present in the vasculature remains unknown. A goal of my laboratory is to elucidate the physiological significance of the sole member of the ankyrin (A) TRP subfamily, TRPA1, in endothelium-dependent regulation of cerebral arteries. TRPA1 is a Ca2+-permeable non-selective cation channel activated by noxious and pungent electrophilic compounds, such as allyl isothiocyanate (AITC), a substance derived from mustard oil and allicin, a compound present in garlic. The structure of the channel is distinguished by the ~14-19 ankyrin repeat (AR) domains present in the intracellular amino terminus. TRPA1 is thought to be involved in diverse physiological processes including nociception, mechanotransduction, and thermal and oxygen sensing. We find that TRPA1 is present in the endothelium of cerebral, but not coronary, mesenteric, renal, or skeletal muscle arteries. Furthermore, AITC-induced activation of TRPA1 causes endothelium-dependent dilation of cerebral arteries. These observations suggest that TRPA1 channels may play a critical but unknown role in the regulation of the cerebral vasculature. Physiological activators of the channel in this tissue are not known. We tested the hypothesis that lipid peroxidation products (LPP) generated by reactive oxygen species produced by NADPH oxidase (NOX) stimulate TRPA1 channels in the endothelium in an autocrine manner. We found that the LPP 4-hydroxy-nonenal (4-HNE) caused concentration-dependent cerebral artery dilation (EC50 = 8.4 µM) that was abolished by the selective TRPA1 antagonist HC-030031. Using total internal reflection fluorescent microscopy (TIRFM) to record TRPA1 Ca2+ sparklets (unitary TRPA1-mediated Ca2+ influx events), we found that the NOX substrate NADPH increased TRPA1 Ca2+ sparklet frequency in primary cerebral artery endothelial cells. This response was blocked by HC-030031. NADPH also dilated isolated and pressurized (80 mmHg) cerebral arteries with pre-developed myogenic tone in a concentration-dependent manner (EC50 = 1.2 µM). Pre-treatment with HC-030031 diminished NADPH-induced dilation, suggesting involvement of TRPA1 channel activity in this response. Significantly, 4-HNE and NADPH-induced dilation were absent from novel, endothelial cell-specific TRPA1 knockout (eTRPA1-/-) mice. Furthermore, immunolabeling studies and proximity ligation assays using primary cerebral artery endothelial cells and isolated cerebral arteries demonstrate co-localization of TRPA1, NOX2, and 4-HNE within myoendothelial junction (MEJ) sites. Together, these data indicate that NOX-derived LPP dilate cerebral arteries by activating TRPA1 in myoendothelial junctions, demonstrating a novel mechanism for the regulation of cerebral blood flow. Our findings suggest that TRPA1 channels may be a suitable target for the development of new therapies for cerebrovascular diseases associated with endothelial dysfunction, such as stroke.