OBJECTIVE - β-Cell response to glucose is characterized by mitochondrial membrane potential (ΔΨ) hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single β-cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of β-cell mitochondria. RESULTS - We show that mitochondria display a wide heterogeneity in ΔΨ and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased ΔΨ heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in ΔΨ of individual mitochondria, ATP-hydrolyzing mitochondria, and uncoupling protein 2 are not significant contributors to ΔΨ heterogeneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity. CONCLUSIONS - We suggest that mitochondrial ΔΨ heterogeneity in β-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target.