VPAC2 receptors sensitive to vasoactive intestinal polypeptide (VIP) and pituitary adenylyl cyclase activating polypeptide (PACAP), PAC1 receptors sensitive to PACAP, and nitric oxide (NO) generation by NO synthase (NOS) are all implicated in cutaneous active vasodilation (AVD) through incompletely defined mechanisms. We hypothesized that VPAC2/PAC1 receptor activation and NO are synergistic and interdependent in AVD and tested our hypothesis by examining the effects of VPAC2/PAC1 receptor blockade with and without NOS inhibition during heat stress. The VPAC2/PAC1 antagonist, pituitary adenylate cyclase activating peptide 6-38 (PACAP6-38) and the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME) were administered by intradermal microdialysis. PACAP6-38, l-NAME, a combination of PACAP6-38 and l-NAME, or Ringer's solution alone were perfused at four separate sites. Skin blood flow was monitored by laser-Doppler flowmetry at each site. Body temperature was controlled with water-perfused suits. Blood pressure was monitored by Finapres, and cutaneous vascular conductance (CVC) calculated (CVC = laser-Doppler flowmetry/mean arterial pressure). The protocol began with a 5- to 10-min baseline period without antagonist perfusion, followed by perfusion of PACAP6-38, l-NAME, or combined PACAP6-38 and l-NAME at the different sites in normothermia (45 min), followed by 3 min of whole body cooling. Whole body heating was then performed to induce heat stress and activate AVD. Finally, 58 mM sodium nitroprusside were perfused at all sites to effect maximal vasodilation for normalization of blood flow data. No significant differences in CVC (normalized to maximum) were found among Ringer's PACAP6-38, l-NAME, or combined antagonist sites during normothermia (P > 0.05 among sites) or cold stress (P > 0.05 among sites). CVC responses at all treated sites were attenuated during AVD (P < 0.05 vs. Ringer's). Attenuation was greater at l-NAME and combined PACAP6-38- and l-NAME-treated sites than at PACAP6-38 sites (P > 0.05). Because responses did not differ between l-NAME and combined treatment sites (P > 0.05), we conclude that VPAC2/PAC1 receptors require NO in series to effect AVD.