Glucagon-like peptide-2 (GLP-2) influences different aspects of the gastrointestinal function, including epithelial growth, digestion, absorption, motility and blood flow. Intraluminal pressure from isolated mouse stomach was recorded in order to investigate if GLP-2 affects gastric tone and to analyze its mechanism of action. Regional differences between diverse parts of the stomach were also examined using circular muscular strips from fundus and antrum. In the whole stomach GLP-2 (0.3 - 100 nM) produced concentration-dependent relaxation with a maximum which was about 75% of relaxation to 1 µM isoproterenol (IC₅₀= 2.5 nM). This effect was virtually abolished by desensitization of GLP-2 receptors or by -chymotrypsin. The relaxant response to GLP-2 was not affected by tetrodotoxin, a blocker of neuronal voltage-dependent Na⁺ channels, but it was significantly reduced by -conotoxin GVIA, a blocker of neuronal N-type voltage-operated Ca²⁺ channels. N-nitro-L-arginine methyl ester, a blocker of nitric oxide synthase, or apamin, a blocker of Ca²⁺ -dependent potassium channels, failed to affect the gastric response to the peptide. However, the relaxation was significantly antagonized by [Lys1,Pro2,5,Arg3,4,Tyr6]VIP^7-28, a vasoactive intestinal peptide (VIP) receptor antagonist (GLP-2 maximum effect = 45% of relaxation to 1 µM isoproterenol) and virtually abolished by desensitization of the VIP receptors. GLP-2 induced concentration-dependent relaxation in carbachol-precontracted fundic strips, but not in antral strips. These results provide the first experimental evidence that GLP-2 is able to induce gastric relaxation acting peripherally on the mouse stomach. The effect appears mediated by prejunctional neural release of VIP and confined to fundic region.