Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic hormone proposed to play a role in both the pathophysiology and treatment of type 2 diabetes. This study has employed the GLP-1 receptor antagonist, exendin-4(9-39)amide (Ex(9-39)) to evaluate the role of endogenous GLP-1 in genetic obesity-related diabetes and related metabolic abnormalities using ob/ob and normal mice. Acute in vivo antagonistic potency of Ex(9-39) was confirmed in ob/ob mice by blockade of the insulin-releasing and anti-hyperglycaemic actions of intraperitoneal GLP-1. In longer term studies, ob/ob mice were given once daily injections of Ex(9-39) or vehicle for 11 days. Feeding activity, body weight, and both basal and glucose-stimulated insulin secretion were not significantly affected by chronic Ex(9-39) treatment. However, significantly elevated basal glucose concentrations and impaired glucose tolerance were evident at 11 days. These disturbances in glucose homeostasis were independent of changes of insulin sensitivity and reversed by discontinuation of the Ex(9-39) for 9 days. Similar treatment of normal mice did not affect any of the parameters measured. These findings illustrate the physiological extrapancreatic glucose-lowering actions of GLP-1 in ob/ob mice and suggest that the endogenous hormone plays a minor role in the metabolic abnormalities associated with obesity-related diabetes. © 2005 Society for Endocrinology.