Glucose dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine Kcells in response to nutrient absorption. In this study we have utilized a specific and enzymatically stable GIP receptor antagonist, (Pro(3))GIP, to evaluate the contribution of endogenous GIP to insulin secretion and glucose homeostasis in mice. Daily injection of (Pro(3))GIP (25 nmol/kg body weight) for 11 days had no effect on food intake or body weight. Nonfasting plasma glucose concentrations were significantly raised (p<0.05) by day 11, while plasma insulin concentrations were not significantly different from saline treated controls. After 11 days, intraperitoneal glucose tolerance was significantly impaired in the (Pro(3))GIP treated mice compared to control (p<0.01). Glucosemediated insulin secretion was not significantly different between the two groups. Insulin sensitivity of 11-day (Pro(3))GIP treated mice was slightly impaired 60 min post injection compared with controls. Following a 15 min refeeding period in 18 h fasted mice, food intake was not significantly different in (Pro(3))GIP treated mice and controls. However, (Pro(3))GIP treated mice displayed significantly elevated plasma glucose levels 30 and 60 min post feeding (p<0.05, in both cases). Postprandial insulin secretion was not significantly different and no changes in pancreatic insulin content or islet morphology were observed in (Pro(3))GIP treated mice. The observed biological effects of (Pro(3))GIP were reversed following cessation of treatment for 9 days. These data indicate that ablation of GIP signaling causes a readily reversible glucose intolerance without appreciable change of insulin secretion.