A glucose oxidase-based glucose microsensor (<10 micro m tip diameter) was used to measure the glucose concentration within single islets under static conditions and during step changes in glucose level. The sensors had response times of 4.1+/-0.5s (n=7) and sensitivities of 8.7+/-1.8 pA/mM (n=11). The sensors performed independent of oxygen up to 15 mM glucose as long as the oxygen level was > 70 mm Hg. Spatially resolved glucose measurements revealed a glucose gradient around and inside single islets. From measurement of the glucose gradient, a glucose consumption rate of 0.48+/-0.14 pmol/nL islet/min (n=6) and an intraislet glucose diffusion coefficient of 3.8 x 10(-7)cm(2)/s were determined. The measurement of the gradient demonstrates that not all cells within an islet in culture are exposed to the same glucose concentration. The sensor was also used to measure the time required for intraislet glucose concentration to reach steady state following a step increase in glucose concentration from 3 to 10 mM at the islet surface. At a depth of 70 micro m inside an islet, glucose reached steady state in 180+/-7s (n=7) for islets with a diameter of 180-220 micro m (smaller islets reach steady-state faster). In the presence of 10 mM mannoheptulose, an inhibitor of glucokinase, the equilibration time was reduced to 122+/-11s (n=6), indicating that glucose utilization by glycolysis limited the time required for glucose to diffuse into the islets. The long times to reach steady state and presence of glucose gradients are important in interpreting data from experiments involving islets in culture.