Oxidative stress is a prominent feature of the placenta in many complications of pregnancy, such as preeclampsia. The cause is primarily unknown, although ischemia-reperfusion injury is one possible mechanism. Our aim was to test this hypothesis by examining the oxidative status of human placental tissues during periods of hypoxia and reoxygenation in vitro. Rapid generation of reactive oxygen species was detected using the fluorogenic probe, 2',7'-dichlorofluorescein diacetate, when hypoxic tissues were reoxygenated. The principal sites were the villous endothelium, and to a lesser extent the syncytiotrophoblast and stromal cells. Increased concentrations of heat shock protein 72, nitrotyrosine residues, and 4-hydroxy-2-nonenal were also observed in the villous endothelial and underlying smooth muscle cells, and in the syncytiotrophoblast. Furthermore, preloading placental tissues with the reactive oxygen species scavengers desferrioxamine and alpha-phenyl-N-tert-butylnitrone reduced levels of oxidative stress after reoxygenation. These changes are consistent with an ischemia-reperfusion injury, and mirror those seen in preeclampsia. Consequently, in vitro hypoxia/reoxygenation may represent a suitable model system for investigating the generation of placental oxidative stress in preeclampsia and other complications of pregnancy.