We evaluated melatonin's antioxidative effect on the free radical-induced impairment of nitric oxide production in the human umbilical artery, which may play an important role in fetal hypoxia and ischemia during preeclampsia. Umbilical artery sections with intact endothelium were obtained from healthy pregnant women who were delivered between 37 and 40 wk of gestation. The production of nitric oxide in the umbilical arteries was stimulated by adding l-arginine followed by incubation for 60 min. Nitric oxide concentrations were estimated by measuring nitrite ions (NO2−), using high-performance liquid chromatography. Prior to the addition of l-arginine, the segments were treated with hydrogen peroxide (H2O2) alone (1, 10, 100 μM), or were pretreated with either 50 mM mannitol or melatonin (20, 100, 500 μM) before adding H2O2. Changes in l-arginine-induced NO2− production were expressed as a percentage of NO2− production at the end of preincubation. NO2− production was significantly increased by incubating the umbilical artery sections with l-arginine (P<0.01). Treatment with H2 O2 significantly reduced l-arginine-induced NO2− production in a concentration-dependent manner (P<0.01). Pretreatment with melatonin significantly increased NO2− production that had been decreased by H2O2 in a concentration-dependent manner (P<0.01). Similarly, pretreatment with mannitol reversed the H2O2-induced reduction in NO2− production (P<0.001). These results indicate that H2O2 may impair nitric oxide synthesis in the endothelium of human umbilical arteries. Melatonin significantly suppresses the H2O2-induced inhibition effect of nitric oxide production, most likely through its ability to scavenge hydroxyl radicals.