1. Acetylcholine-mediated (ACh-mediated) secretion of fluid and protein from rat submandibular glands was enhanced by intravenous injection of vasoactive intestinal peptide (VIP) and of secretin but not of peptide histidine isoleucine (PHI) or gastric inhibitory peptide (GIP). 2. When VIP and ACh were administered together, the enhancement of fluid secretion was inhibited by pretreatment with atropine or 4-DAMP and the enhancement of protein secretion was inhibited by pretreatment with atropine or phentolamine. 3. The enhancement of the ACh-induced secretion of fluid by secretin was strongly inhibited by pretreatment with atropine, and it was weakly inhibited by pretreatment with phentolamine or haloperidol. 4. These results suggest that the synergistic effects of VIP, PHI, secretin and GIP on the ACh-mediated secretion of fluid and protein from the rat submandibular gland do not reflect the extent of the structural homology of each peptide to VIP.