Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic β-cells induced by glucagon-like peptide 1 (7–36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2–100 nM). Exendin (9–39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from –90 to –50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K⁺ channel (K_ATP). This GLP-1 effect was prevented by exendin (9–39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through K_ATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on K_ATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of K_ATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced K_ATP activity. The half-maximal concentrations (k _i) of ATP were 11.6 µM without and 5.6 µM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces K_ATP activity by elevating the sensitivity of K_ATP to ATP, resulting in depolarization of pancreatic β-cells. This GLP-1 action is independent of the cAMP signalling pathway.