Studies of dispersed β cells have been used to infer their behavior in the intact pancreatic islet. When dispersed, β 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 β cells. However, previously reported synchronous and uniform Ca2+ activity and electrical responses indicate that β cell behavior within intact islets is more uniform. Therefore, uncertainty remains whether β 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 β cells and within intact islets. Over 90% of β cells in intact islets responded to glucose with significantly elevated NAD(P)H levels, compared with less than 70% of dispersed β cells. In addition, all responding β cells within intact islets exhibited a sigmoidal glucose dose response behavior with inflection points of ~8 mM glucose. These results suggest that β cell heterogeneity may be functionally less important in the intact islet than has been predicted from studies of dispersed β cells and support the role of glucokinase as the rate-limiting enzyme in the β cell glucose response.