The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between β cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gh(lox)). Analysis of AdenoCre-treated islets indicated that the gk(lox) allele in ~30% of islet cells was converted to a nonexpressing variant (gk(del)). This resulted in a heterogeneous population of cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet β cells from gk(lox/lox) mice. In AdenoCre-infected islets, approximately one-third of the β cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low- responding cells were not sigmoidal and reached a maximum at ~5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an K(cat)S0.5 of α8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in β cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.