Pituitary adenylate cyclase activating peptide (PACAP), a recently described vasoactive intestinal peptide-like neuropeptide, was found to be present in neurons in the submandibular gland of the ferret, where PACAP-immunoreactive nerve fibers were distributed around blood vessels, acini and ducts. Most of the PACAP-immunoreactive fibres were distinct from those storing vasoactive intestinal peptide. PACAP occurs in tissues as PACAP1-38 and PACAP1-27. PACAP1-38 and PACAP1-27 but not PACAP16-38 displayed biological activity with about the same potency. They exerted vasodilator effects on the submandibular vasculature, which resulted in a greater fall in vascular resistance than an equimolar dose of vasoactive intestinal peptide. The vasodilator response was independent of muscarinic receptor activation. Neither vasoactive intestinal peptide nor PACAP alone evoked any flow of saliva. However, both vasoactive intestinal peptide and PACAP enhanced the fluid response to acetylcholine, and the flow of saliva as well as the output of protein in response to parasympathetic nerve stimulation, vasoactive intestinal peptide being more potent than PACAP. In vitro, protein was released from submandibular gland tissue in response to both vasoactive intestinal peptide and PACAP, vasoactive intestinal peptide being more potent than PACAP. PACAP (and vasoactive intestinal peptide) exerted its in vitro effect following adrenoceptor and muscarinic blockade and following degeneration of sympathetic nerves. Sympathetic denervation combined with parasympathetic preganglionic denervation resulted in supersensitivity to both vasoactive intestinal peptide and PACAP. The fact that PACAP and vasoactive intestinal peptide occur in different nerve fibre populations suggests different roles for the two peptides in the submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)