Preeclampsia is a hypertensive complication of human pregnancy characterized by generalized maternal endothelial cell activation. Circulating pro-inflammatory cytokines derived from the placenta are thought to play a key role. We recently demonstrated that hypoxia-reoxygenation (H/R) of placental tissues in vitro causes equivalent oxidative stress to that seen in preeclampsia. Our aim was to determine whether H/R also increases production of tumor necrosis factor-α (TNF-α), and whether conditioned media from samples exposed to H/R causes activation of human umbilical vein endothelial cells (HUVECs). Concentrations of mRNA encoding TNF-α were significantly higher in placental tissues subjected to H/R compared to hypoxic or normoxic controls. Although there was no difference in the concentrations of TNF-α protein in tissue homogenates, levels of TNF-α protein in the medium were significantly higher after H/R compared to controls, indicating increased secretion. Furthermore, conditioned medium from samples subjected to H/R caused increased expression of E-selectin by HUVECs, and the addition of anti-TNF-α antibodies significantly reduced that activation. These results are consistent with our hypothesis that intermittent perfusion of the placenta, secondary to reduced trophoblast invasion, causes increased secretion of TNF-α, and that this contributes to the activation of maternal endothelial cells that characterizes preeclampsia.