Glucose metabolism via the pentose cycle, glycolysis and the Krebs cycle was quantified in bovine microvascular endothelial cells. The major measured end-product of glucose was L-lactate, with relatively small amounts of glucose carbons converted to CO2 and pyruvate. The pentose cycle accounted for less than 4% of the glucose utilized. About 60-70% of the metabolized glucose carbons could not be accounted for by lactate, pyruvate and CO2. Insulin stimulated glycolysis and pentose cycle activity, but had no effect on glucose oxidation via the Krebs cycle. As the pentose cycle is a major source of NADPH which is required for the synthesis of nitric oxide (the endothelium relaxing factor), insulin may play a role in regulating NO generation in endothelial cells by modulating the pentose cycle activity.