The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (KATP) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic β-cells. Membrane potential, single channel and whole-cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic β-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic β-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 µM) completely reversed the inhibitory actions of GLP-1 on the membrane KATP conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP-1 and its restoration by 20 µM W-7 or 10 µM calmidazolium at the single channel level. Bath application of 20 µM W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic β-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system. Experimental Physiology (2001) 86.3, 331-339.