Glucagon-like peptide-1 (GLP-1) is an incretin hormone, released from intestinal L-cells in response to food ingestion, which stimulates insulin release and improves glycaemia in type 2 diabetes. Drugs based on mimicking the action of GLP-1 or inhibiting clearance of the endogenous hormone have been licensed recently for the treatment of type 2 diabetic subjects. The idea of therapeutically targeting L-cells to enhance endogenous release of GLP-1 and Peptide YY is increasingly popular, but is hampered by current uncertainties surrounding normal L-cell function. To enable single cell analysis of primary murine L-cells, we generated transgenic mice expressing a yellow fluorescent protein under the control of the proglucagon promoter. These mice exhibit yellow fluorescence in L-cells, pancreatic alpha cells, and populations of brainstem neurones. L-cells, purified from the transgenic mice by flow cytometry and analysed by quantitative RT-PCR, expressed high levels of K-ATP channel subunits, glucokinase and sodium glucose cotransporter 1 (SGLT1). The expression levels of Kir6.2, SUR1 and glucokinase were similar to those found in pancreatic alpha and beta cells. Single L-cells in primary culture, identified by their yellow fluorescence, were electrically active, and stimulated by glucose and tolbutamide. Identified L-cells loaded with fura-2 exhibited intracellular calcium rises in response to application of glucose, alpha-methylglucose or tolbutamide. In mixed primary cultures of upper small intestine or colon, GLP-1 secretion was glucose-dependent with an EC50 in the low millimolar range, and was also stimulated by tolbutamide or alpha-methylglucose. The combined results from expression analysis, electrophysiological recordings, single cell fluorescence calcium imaging and GLP-1 secretion implicate important roles for K-ATP channels and SGLT1 in L-cell glucose-sensing.