Electrical vagal stimulation increased immunoreactive peptide histidine isoleucine (IR-PHI) and vasoactive intestinal polypeptide (IR-VIP) levels in the portal plasma in anesthetized dogs, depending on the stimulation frequency. Atropine failed to suppress the vagal release of IR-PHI and IR-VIP, whereas hexamethonium abolished the increase of both peptide immunoreactivities. Gel filtration profiles of IR-PHI and IR-VIP in the portal plasma obtained during vagal stimulation revealed major peaks of IR-PHI and IR-VIP eluting in the same positions as synthetic PHI-27 and VIP-28, respectively. These data demonstrate that PHI and VIP are coreleased by vagal stimulation via a nicotinic ganglionic mechanism. An additional peak of larger molecular weight IR-PHI was observed in gel filtration of the portal plasma obtained under higher frequency vagal stimulation, when measured by the N-terminal-specific PHI antiserum. The presence of this larger form of IR-PHI together with a PHI-27-like component was also demonstrated in tissue extracts of the entire length of the gastrointestine, particularly in higher concentration in the stomach. The structure and the physiological significance of this larger form of IR-PHI remains to be elucidated.