The parasympathetic nervous system is probably involved in migraine pathogenesis. Its activation releases a mixture of signalling molecules including vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), which subsequently stimulate VPAC1, VPAC2 and PAC1 receptors. The objective of the present study was to investigate the in vivo effect of VIP, PACAP-27, PACAP-38, the selective VPAC1 agonist ([Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27)) and a PAC1 agonist, maxadilan on rat middle meningeal artery (MMA) diameter using the closed cranial window model. Selective antagonists were used for further characterization of the responses. Reverse transcriptase-polymerase chain reaction experiments were also conducted to determine expression of mRNA of PACAP receptors in the MMA. The results showed that VIP, PACAP-38, PACAP-27 and the VPAC1 specific agonist evoked significant dilations with the rank order of potency; VIP = PACAP-38 > PACAP-27 = [Lys15, Arg16, Leu27]-VIP(1-7)-GRF(8-27). Significant inhibition of dilation was only observed for the VPAC1 antagonist PG97-269 on PACAP-38-induced dilation of MMA. The VPAC2 antagonist PG99-465 and PAC1 antagonist PACAP(6-38) did not significantly block VIP- or PACAP-induced dilation. Expression of mRNA of all three receptors was detected in the MMA. In conclusion, the VPAC1 receptor seems to be predominant in mediating MMA dilation. A selective VPAC1 antagonist may be a candidate molecule in the treatment of migraine headache.