Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to carbohydrate and fat in the diet. Despite the interest in GLP-1 as an antidiabetic agent, very little is known about the mechanism of stimulus-secretion coupling in L-cells. We investigated the electrophysiological events underlying glucose-induced GLP-1 release in the GLP-1-secreting cell line, GLUTag. Cells were studied using perforated-patch and standard whole-cell patch clamp recordings. GLUTag cells were largely quiescent and hyperpolarized in the absence of glucose. Increasing the glucose concentration between 0 and 20 mmol/l decreased the membrane conductance, caused membrane depolarization, and triggered the generation of action potentials. Action potentials were also triggered by tolbutamide (500 micro mol/l) and were suppressed by diazoxide (340 micro mol/l) or the metabolic inhibitor azide (3 mmol/l), suggesting an involvement of K(ATP) channels. Large tolbutamide-sensitive washout currents developed in standard whole-cell recordings, confirming the presence of K(ATP) channels. RT-PCR detected the K(ATP) channel subunits Kir6.2 and SUR1 and glucokinase. GLP-1 secretion was also stimulated by glucose over the concentration range 0-25 mmol/l and by tolbutamide. Our results suggest that glucose triggers GLP-1 release through closure of K(ATP) channels and action potential generation.