To determine whether hyperglycemia-induced increase in oxidative burst undergoes adaptive changes, the time course of superoxide (SO) production in human umbilical vein endothelial cells treated with 13.75 mM (D250) or 27.5mM dextrose (D500) was measured using the hydroethidine (HE) fluorescence method. The rate of SO production (mean +/- S.D., in arbitrary units) in cells treated with D500 during the first hour (0.758 +/- 0.367) or with D250 (0.618 +/- 0.126) was significantly higher than the rate observed in control cells treated with 100mg/dl dextrose, (D100; 0.474 +/- 0.125) (P < 0.001). However, the rate of SO production during the second, third, fourth, and fifth hour was not significantly different from that measured in control cells. The fluorescence at baseline for control cells was 3.4 +/- 2.3 and for cells treated with D500 for 1, 2, 3, 4, and 5h was 3.4 +/- 1.9, 15.2 +/- 2.5, 21.6 +/- 2.3, 27.4 +/- 3.4, and 31.8 +/- 4.3 respectively (P < 0.001). The increased baseline fluorescence suggests that the antioxidant pool may be depleted within the first hour of exposure to high concentrations of dextrose. The latter possibility is supported by the observation that treatment of cells with varying concentrations of ascorbate (15, 150, and 1500 microM) or alpha-tocopherol (10,100 and 1000 microM) prevents D500 induced increase in SO production. It is concluded that increased oxidative load in sustained chronic hyperglycemia is probably the result of depletion of antioxidant pool rather than secondary to sustained increase in SO production.