Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced from arachidonic acid by cytochrome P450 (CYP) epoxygenases (EPOXs) and metabolized to vicinal diols by soluble epoxide hydrolase (sEH). In brain, EETs are produced by astrocytes and vascular endothelium and are involved in the control of cerebral blood flow (CBF). Recent evidence, however, suggests that EPOX and sEH are present in perivascular vasodilator nerve fibers innervating the cerebral surface vasculature. Herein we tested the hypothesis that EETs are a nerve-derived relaxing factor in the cerebral circulation. We first traced these fibers by retrograde labeling in the rat to the trigeminal (TG) and sphenopalatine ganglia (SPG). We then examined expression of CYP EPOXs and sEH in these ganglia. RT-PCR and western blot identified CYP2J3 and CYP2J4 EPOX isoforms and sEH in both TG and SPG, and immunofluorescence double labeling identified CYP2J- and sEH-immunoreactivity in neuronal cell bodies of both ganglia. To evaluate the functional role of EETs in neurogenic vasodilation, we elicited cortical hyperemia by electrically stimulating efferent cerebral perivascular nerve fibers and by chemically stimulating oral trigeminal fibers with capsaicin. Cortical blood flow responses were monitored by laser-Doppler flowmetry (LDF). Local administration to the cortical surface of the putative EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 30 µmol/L) attenuated the CBF responses to electrical and chemical stimulation. These results suggest that EETs are produced by perivascular nerves and play a role in neurogenic vasodilation of the cerebral vasculature. The findings have important implications to such clinical conditions as migraine, vasospasm after subarachnoid hemorrhage, and stroke.