Studies of dispersed beta cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, beta cells exhibit multiple metabolic glucose-response populations with different insulin secretion properties. This has led to a model for glucose-dependent insulin secretion from the islet based on a step-wise recruitment of individual beta cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that beta cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether beta cell metabolic heterogeneity is functionally important in intact islets. We have used two-photon excitation microscopy to measure and compare the glucose-induced NAD(P)H autofluorescence response in dispersed beta cells and within intact islets. Over 90% of beta cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed beta cells. In addition, all responding beta cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of approximately 8 mm glucose. These results suggest that beta cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed beta cells and support the role of glucokinase as the rate-limiting enzyme in the beta cell glucose response.