Endocannabinoid lipids are known to exert orexigenic effects via central cannabinoid CB1 and CB2 receptors, which have also been identified in islet endocrine cells. However, there is no consensus on whether the receptors are expressed by beta-cells, nor what effect CB1 and CB2 receptor agonists have on insulin secretion. In the current study we have therefore used the mouse MIN6 beta-cell line rather than primary islets, which are heterogeneous clusters of endocrine cells. Cannabinoid receptor and diacylglycerol lipase isoform mRNAs were detected in MIN6 cells by RT-PCR and immunocytochemistry was used to identify cannabinoid receptor expression by MIN6 cells. Changes in cyclic AMP and intracellular calcium were measured by immunoassay and microfluorimetry, respectively, and insulin secretion from perifused MIN6 pseudoislets was determined by radioimmunoassay. MIN6 beta-cells express the cannabinoid synthesising enzyme diacylglycerol lipase and CB1 and CB2 receptors, which are coupled to inhibition of beta-cell cyclic AMP generation and stimulation of intracellular calcium levels. Cannabinoid receptor activation with pharmacological agonists resulted in reversible elevations in insulin secretion at both 2 mM and 20 mM glucose. Synthesis of endocannabinoids by beta-cells may provide an additional mechanism for stimulation of insulin secretion through activation of beta-cell CB1 and/or CB2 cannabinoid receptors.