Insulin production by the pancreatic islet is tightly coupled to the concentration of blood glucose. The mechanism by which glucose controls proinsulin biosynthesis in beta cells is poorly understood. Analysis of insulin gene expression in individual cells within whole, living islets using adenovirus gene transfer and direct observation of insulin promoter-directed green fluorescent protein activity indicates that beta cells are functionally heterogeneous. An increase in glucose concentration not only stimulates expression within individual beta cells, but unexpectedly acts to increase the total number of positive cells. The net islet response to a given glucose stimulus reflects an integrated action of beta cells with individually differing behaviors. This additional level of functional complexity may provide new insights into the pathophysiology and treatment of diabetes mellitus.