We investigated the contribution of perivascular nerves and neurotransmitters to cortical spreading depression (CSD)-associated hyperperfusion in the rat. Chronic transection of the nasociliary nerve (NCN, 2 wk before) decreased ipsilateral CSD-associated hyperperfusion by 23 ± 13% (mean ± SD; n = 5, P < 0.05), whereas acute transection of the NCN or sham surgery had no effect (n = 8). When the NCN and parasympathetic nerve fibers (PSN) were both chronically transected, CSD hyperperfusion was attenuated by 55 ± 19% (n = 5, P < 0.05). Cerebrovascular reactivity to hypercapnia was not significantly affected. Brain topical superfusion of the muscarinic receptor antagonist atropine (10⁴ M) caused a reduction of CSD hyperperfusion by 41 ± 13% (n = 5, P < 0.05). The competitive blockade of calcitonin gene-related peptide (CGRP) receptors by CGRP-(837) (5 × 10⁷ M) afforded a decrease by 49 ± 19% (n = 5, P < 0.05), without affecting CO₂ reactivity (n = 4). The combined application of both CGRP-(837) and atropine further attenuated CSD hyperperfusion (by 69 ± 17%, n = 5, P < 0.05). After chronic NCN and PSN transection brain topical superfusion of CGRP-(837) (5 × 10⁷ M) reduced CSD hyperperfusion slightly by 9.5 ± 5% (n = 3). Atropine (10⁴ M) afforded a decrease by 17 ± 6% (n = 3). These reductions were not statistically significant. We conclude that CSD-associated hyperperfusion is mediated in part by a depolarization of trigeminal sensory and parasympathetic nerve fibers, resulting in a release of vasoactive trigeminal and parasympathetic neurotransmitters.