objective: The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC, VPAC and VPAC) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. methods: Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. results: PACAP, but not VIP, potently stimulated cAMP, IP accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC and VPAC receptor-like immunoreactivity appeared to be distinct in the spinal cord. conclusions: The pharmacological profile in the spinal cord suggested that a PAC receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC and VPAC receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.