Abstract A flow-injection system for the enzymatic assay of glucose is described. It utilizes double injection of sample and enzyme [glucose oxidase (GOD), ca. 500 U ml −1] or mixed enzyme [GOD, ca. 500 U ml −1; horseradish peroxidase (POD), ca. 220 U ml −1) into an air-saturated phosphate carrier stream (pH 7.0). The reaction product(s) were monitored amperometrically in a three-electrode wall-jet microflow cell (4 μl). Product hydrogen peroxide analysis by pulsed amperometric detection was compared with analysis using the electrode mediators 2,2′-azinobis(3-ethylbenz- thiazoline-6-sulfonic acid) (ABTS) and potassium hexacyanoferrate(II). The last method was favored, coupled with oxygen supplementation achieved by use of a gas-permeable silicone-rubber tube reaction coil. This provided a useful curvilinear response to > 14 mM glucose, with a linear response to ca. 7 mM; the relative standard deviations for 3.0 and 0.3 mM glucose were 1.3% and 1.8%, respectively, and the detection limit was 0.01 mM glucose using stopped-flow. Both sensitivity and linearity were improved significantly by the use of stopped-flow techniques.