In the rat gastric fundus, vasoactive intestinal polypeptide (VIP) contributes to the nonadrenergic noncholinergic (NANC) inhibitory neurotransmission and coexists with its related peptide, peptide histidine isoleucine (PHI). As the two neuropeptides are co-released and have similar actions in different biological systems, the effect of PHI in the rat gastric fundus was studied in order to investigate whether it might be a co-transmitter of VIP in the inhibitory NANC neurotransmission in this tissue. Auxotonic responses were measured in longitudinal muscle strips from the gastric fundus of reserpinized rats (5 mg/kg i.p., 24 hr before sacrifice), suspended between parallel platinum electrodes in Krebs solution containing atropine (1 microM) and 5-hydroxytryptamine (3 microM). PHI induced concentration-dependent relaxations (10 nM-1 microM), qualitatively similar to those induced by electrical field stimulation (1 msec, supramaximal voltage, 0.25-16 Hz) and by VIP (0.3-100 nM), although PHI was about 30 times less potent than VIP. The submaximal relaxation induced by PHI (100 nM) was not influenced by tetrodotoxin (1 microM), hexamethonium (500 microM) and propranolol (10 microM) plus phentolamine (3 microM). Incubation during 60 min with a specific PHI-antiserum completely prevented the relaxation induced by PHI (100 nM) and reduced by 26% that induced by electrical field stimulation at 1 Hz; the procedure had no influence on the relaxation induced by VIP (3 nM) or isopropylnoradrenaline (10 nM). Incubation during 60 min with a specific VIP-antiserum completely prevented the relaxation induced by VIP (3 nM) and halved the relaxatory response to electrical field stimulation at 1 Hz, while it had no influence on the relaxation induced by PHI (100 nM) or isopropylnoradrenaline (10 nM). Control serum had no influence on any of the relaxant stimuli. In conclusion, PHI mimics the electrically induced NANC relaxation in the rat gastric fundus; its action is located at muscle level and does not involve alpha-, beta- or nicotinic receptors. The results obtained in the presence of the antisera provide further confirmation that VIP contributes to the NANC inhibitory neurotransmission of the rat gastric fundus and leave open the possibility that PHI is involved as co-transmitter.