Context: Glucagon-like peptide-1 (GLP-1) secretion from L-cells and postprandial inhibition of gastrointestinal motility. Objective: Investigate whether physiological plasma concentrations of GLP-1 inhibit human postprandial motility and determine mechanism of action of GLP-1 and analog ROSE-010 action. Design: Single-blind parallel study. Setting: University hospital laboratory. Participants: Healthy volunteers investigated with antroduodenal manometry. Human gastric and intestinal muscle strips. Interventions: Motility indices (MIs) obtained before and during GLP-1 or saline infusion. Plasma GLP-1 and glucagon-like peptide-2 (GLP-2) measured by radioimmunoassay. Gastrointestinal muscle strips investigated for GLP-1- and ROSE-010-induced relaxation employing GLP-1 and GLP-2 and their receptor localization, and blockers exendin(9-39) amide, L-omega-nitro-monomethylarginine (L-NMMA), 2',5'-dideoxyadenosine (DDA), and tetrodotoxin (TTX) to reveal target mechanism of GLP-1 action. Main Outcome Measures: Postprandial gastrointestinal relaxation by GLP-1. Results: In humans, food intake increased MI to 6.4 +/- 0.3 (antrum), 5.7 +/- 0.4 (duodenum), and 5.9 +/- 0.2 (jejunum). GLP-1 administered intravenously raised plasma GLP-1, but not GLP-2. GLP-1 0.7 pmol/kg/min suppressed corresponding MI to 4.6 +/- 0.2, 4.7 +/- 0.4, and 5.0 +/- 0.2, whereas 1.2 pmol/kg/min suppressed MI to 5.4 +/- 0.2, 4.4 +/- 0.3, and 5.4 +/- 0.3 (P < 0.0001 to 0.005). In vitro, GLP-1 and ROSE-010 prevented contractions by bethanechol and electric field stimulation (P < 0.005 to 0.05). These effects were disinhibited by exendin(9-39) amide, L-NMMA, DDA, or TTX. GLP-1 and GLP-2 were localized to epithelial cells, GLP-1 also at myenteric neurons. GLP-1R and GLP-2R were localized at myenteric neurons but not muscle. Conclusions: GLP-1 and ROSE-010 inhibit postprandial gastrointestinal motility through GLP-1R at myenteric neurons, involving nitrergic and cyclic adenosine monophosphate-dependent mechanisms.