1. The localization, tissue concentrations, and effects of pituitary adenylate cyclase activating peptide (PACAP) 27 and 38 were investigated in cat and human lower oesophageal sphincter (LOS), and compared with those of vasoactive intestinal peptide (VIP) and helospectin. 2. PACAP-immunoreactive nerve structures were found in the cat and human LOS, with an abundance in the circular smooth muscle layer. PACAP 27-immunoreactivity was often co-localized with VIP-immunoreactivity. 3. In cat tissue, PACAP (PACAP 27 plus PACAP 38) concentrations were 50 fold lower than VIP concentrations; in human tissue they were 10 fold lower. 4. PACAP 27, PACAP 38, helospectin I, and VIP induced concentration-dependent relaxations in circular smooth muscle preparations from cat and human LOS. The order of potency was: VIP > helospectin I > or = PACAP 27 > PACAP 38. NG-nitro-L-arginine, scopolamine, or apamin, did not influence the relaxant effects of PACAP 27 or VIP. 5. In cat preparations, both cyclic AMP and cyclic GMP levels were increased after exposure to PACAP 27 and helospectin I, whereas exposure to VIP was followed by an increase in cyclic AMP levels only. In human preparations, there was an increase in cyclic AMP levels without any change in cyclic GMP levels. 6. These results suggest that in the cat and human LOS, PACAP 27 and VIP can occur within the same nerve structures. PACAP 27 has a potent relaxant action, but its functional importance has to be established.