Background and Purpose The pituitary adenylate cyclase‐activating peptide (PACAP) family is of clinical interest for the treatment of migraine. These peptides activate three different PACAP‐responsive class B G protein‐coupled receptors: the PAC1, VPAC1 and VPAC2 receptors. The PAC1 receptor may be alternatively spliced, generating variants that can differ in their pharmacological or signalling profiles. To inform drug discovery efforts targeting migraine, we need to better understand how the different PACAP‐responsive receptors signal and how effectively these responses can be blocked by antagonists. Experimental Approach The signalling profiles of the human PAC1n, PAC1s, VPAC1 and VPAC2 receptors were examined in transfected Cos7 cells for cAMP, IP1, pAkt, pERK and pCREB. Biased signalling was then quantified. The ability of antagonists to block PACAP‐38, PACAP‐27 or VIP stimulated cAMP accumulation at PACAP‐responsive receptors was also determined. Key Results PACAP‐responsive receptors exhibited varied pharmacological profiles but activated signalling in a similar manner. The PAC1n and PAC1s receptors displayed distinct pharmacology. At the PAC1s receptor, VIP and PHM were more potent than at the PAC1n receptor. PACAP‐responsive receptors displayed agonist‐dependent antagonism where PACAP‐38 was less effectively antagonised compared to PACAP‐27 and VIP. Conclusions and Implications The distinct pharmacological profile displayed by the PAC1s receptor suggests that it can act as a dual receptor for VIP and PACAP. Furthermore, the effectiveness of blocking a signalling pathway can be influenced by which endogenous PACAP family agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PACAP system. LINKED ARTICLES This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc