Lipids have been implicated in β-cell stimulus-secretion coupling. Thus, lipases in β-cells would be required to generate coupling factors from intracellular lipids. Indeed, we found that glucose stimulates lipolysis in rodent islets and clonal β-cells. Lipolysis and diglyceride lipase activity in islets are abolished by orlistat, a pan-lipase inhibitor. Moreover, orlistat dose-dependently inhibits glucose- and forskolin-stimulated insulin secretion, while leaving glucose oxidation and the rise in ATP-to-ADP ratio intact. In an effort to identify β-cell lipase(s), we found that hormone-sensitive lipase (HSL), the rate-limiting enzyme for acylglyceride hydrolysis in adipocytes, is active in rodent β-cells. To further address the role of HSL, a global and β-cell–specific inactivation, respectively, of the lipase has been created in mice. Whereas our line of HSL null mice is moderately glucose intolerant due to reduced peripheral insulin sensitivity, it exhibits normal islet metabolism and insulin secretion. Preliminary analysis of the β-cell–specific HSL knockout has revealed no evidence for disturbed islet function. Thus, studies of ours and others indicate that there is a complex lipid regulatory component in β-cell stimulus-secretion coupling. The role of HSL and other lipases needs to be further clarified to provide a balanced view of the role of lipids and lipolysis in β-cells.