To test the hypothesis that endogenous neuropeptide Y (NPY) counteracts the vasodilator effects of calcitonin gene-related peptide (CGRP), we used isolated mesenteric resistance arteries of rats and mice. With immunohistochemistry, we observed CGRP-containing fibers along and in the vicinity of a subset of NPY- or tyrosine hydroxylase-immunoreactive fibers. The CGRP1 receptor component calcitonin-related-like receptor was expressed by periarterial nerves and smooth muscle cells, whereas receptor activity-modifying protein 1 was observed primarily on the smooth muscle. In organ chambers, exogenous CGRP caused relaxations that were reversed by exogenous NPY. The effects were inhibited by 1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]-carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)-methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS, a CGRP1 receptor antagonist; pK(B) = 8.54 +/- 0.52) and (R)-NZ-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226, a Y1 antagonist; pK(B) = 7.00 +/- 0.49), respectively. Pretreatment with capsaicin (1 muM; 20 min) and the presence of BIBN4096BS (20 nM) increased contractile responses to K(+) (20-40 mM) and electrical field stimulation (EFS; 1-32 Hz). NPY increased contractile responses to K(+) and BIBP3226 (400 nM) reduced contractile responses to EFS. These effects were inhibited by capsaicin and BIBN4096BS, respectively. Furthermore, the relaxing effect of exogenous CGRP (10 nM) during phenylephrine-induced contraction (30 muM) was reversed by EFS, and this effect was reduced in the presence of BIBP3226. We confirmed that bioactive concentrations of endogenous CGRP and NPY can be released from periarterial sensory-motor and sympathetic nerves, respectively, and we demonstrate for the first time functional antagonism between endogenous NPY and CGRP at the level of the smooth muscle.