We reported that nicotine stimulates presynaptic nicotinic acetylcholine (ACh) receptors in adrenergic nerves and activates TRPV1 located on CGRP-containing vasodilator nerves (CGRPergic nerves) resulting in vasodilation. The aim of this study is to investigate whether proton acts as a neuromodulator for perivascular CGRPergic nerves. Rat perfused mesenteric vascular beds without endothelium were contracted by perfusion with Krebs solution containing methoxamine and the pH levels of outflowed perfusate were measured with a pH meter. ACh at high concentrations lowered pH levels of the perfusate concomitant with vasodilation. Capsaicin (high concentration), ruthenium red and atropine but not guanethidine inhibited ACh-induced lowering of pH levels and vasodilation. Mecamylamine blunted atropine-resistant ACh responses. Capsaicin injection at low concentrations induced vasodilation and pH lowering, which were abolished by ruthenium red. HCl injection induced vasodilation and pH lowering of the perfusate. HCl-induced vasodilation, but not pH lowering, was inhibited by cold-storage denervation (4°C for 72 h), high concentration capsaicin and ruthenium red. The study using a fluorescent pH indicator demonstrated perivascular pH decrease after capsaicin, ACh or nicotine applied outside small mesenteric arteries. Immunohistochemical study showed dense innervation of adrenergic tyrosine hydroxylase (TH)-like immunoreactivity (LI) and CGRP-LI-containing nerves and both appeared in the same neuron. CGRP-LI and TRPV1-LI-containing nerves also appeared in the same neurons. These results suggest that excitement of CGRPergic nerves releases protons to activate presynaptic TRPV1 in CGRPergic nerves and thereby facilitate neurotransmission as positive feedback modulator.