Nicotine stimulates presynaptic nicotinic acetylcholine receptors in perivascular adrenergic nerves and releases unknown transmitter(s) that activate transient receptor potential vanilloid-1 (TRPV1) located on calcitonin gene-related peptide (CGRP)-containing (CGRPergic) nerves, resulting in vasodilation. The present study investigated a potential transmitter transmitting between perivascular adrenergic nerves and CGRPergic nerves. Rat mesenteric vascular beds without endothelium were contracted by perfusion with Krebs' solution containing methoxamine, and the perfusion pressure and pH levels of the perfusate were measured. Nicotine perfusion for 1 min induced concentration-dependent vasodilation and lowered pH levels, which were abolished by cold-storage denervation of preparations, guanethidine (adrenergic neuron blocker), and mecamylamine (nicotinic alpha(3)beta(4)-acetylcholine receptor antagonist). Capsazepine (TRPV1 antagonist) blunted nicotine-induced vasodilation, but had no effect on the reduction of pH. Injection of hydrochloric acid (HCl) and perfusion of Krebs' solution at low pH (6.0-7.2) induced vasodilation. HCl-induced vasodilation was inhibited by cold-storage denervation, capsazepine, capsaicin (CGRP depletor), and CGRP(8-37) (CGRP receptor antagonist). Perfusion of adrenergic transmitter metabolites (normetanephrine and 3-methoxydopamine), but not of other metabolites, induced vasodilation, which was not inhibited by capsaicin treatment. Immunohistochemical staining of mesenteric arteries showed dense innervation of CGRP- and TRPV1-immunopositive nerves, with both immunostainings appearing in the same neuron. Mesenteric arteries were densely innervated by neuropeptide Y-immunopositive nerves, which coalesced with CGRP-immunopositive nerves. Scanning and immunoscanning electron microscopic images showed coalescence sites of different perivascular fibers before they intruded into smooth muscles. These results indicate that nicotine initially stimulates adrenergic nerves via nicotinic alpha(3)beta(4)-receptors to release protons and thereby induces CGRPergic nerve-mediated vasodilation via TRPV1.