Previous studies have shown that inhibitory transmission in guinea pig stomach involves an interplay between vasoactive intestinal peptide (VIP) and nitric oxide (NO). The present study examined the contribution of pituitary adenylate cyclase-activating peptide (PACAP), a homologous peptide present in gastric and intestinal myenteric neurons. VIP, PACAP-27 and PACAP-38 induced concentration-dependent relaxation that was partly inhibited by the antagonists VIP10-28 and PACAP6-38 and the NO synthase inhibitor NG-nitro-L-arginine (L-NNA). Only relaxation induced by PACAP-27 and PACAP-38 was partly inhibited by apamin. Electrical field stimulation (0.25-16 Hz) induced frequency-dependent relaxation and PACAP release (maximum of 35.7 fmol/100 mg-min or 7-fold above basal levels). Electrical field stimulation-induced relaxation was partly inhibited by a combination of selective monoclonal antibodies to PACAP-27 and PACAP-38 (42 +/- 7% at 16 Hz) and by the antagonists VIP10-28 (29 +/- 9%) and PACAP6-38 (29 +/- 3%). The relaxation was also partly inhibited by L-NNA (51 +/- 12% at 16 Hz) and apamin (36 +/- 4%). The effects of a combination of apamin and L-NNA were additive, amounting to 75 +/- 3% inhibition. The effect of L-NNA reflected inhibition of NO release from nerve terminals, as well as NO generation in muscle cells by the action of VIP and PACAP; the effect of apamin reflected blockade of the action of PACAP. Thus, inhibitory transmission in guinea pig gastric fundus represents the combined actions of VIP, PACAP and NO released from nerve terminals and NO generated in muscle cells. The postjunctional actions of PACAP are mediated by a VIP/PACAP-II receptor and by a PACAP-specific, apamin-sensitive receptor.