The experiments described in this paper were designed to determine the mechanism underlying the increase in 8-isoprostaglandin F2α (8-epi-PGF2α) production by cultured human endothelial cells during reoxygenation following hypoxia. Human umbilical artery endothelial cells were grown on microcarrier beads and exposed to sequential periods of normoxia, hypoxia, and reoxygenation. The amount of 8-epi-PGF2α in the medium was determined by ELISA. The production of 8-epi-PGF2α decreased by greater than 90% during hypoxia. Upon reoxygenation 8-epi-PGF2α production increased linearly for 90 min reaching nearly 3 times normoxic levels. When added to the medium during reoxygenation, neither superoxide dismutase nor Tiron, a cell-permeable superoxide scavenger, inhibited 8-epi-PGF2α production. However, 8-epi-PGF2α production was inhibited by catalase. The production of 8-epi-PGF2α was also inhibited by indomethacin and aspirin. Exogenous hydrogen peroxide stimulated 8-epi-PGF2α production by normoxic cells, and aspirin inhibited the hydrogen peroxide-mediated increase in 8-epi-PGF2α production. These results indicate that the reactive oxygen species responsible for 8-epi-PGF2α synthesis during reoxygenation is hydrogen peroxide and that in endothelial cells 8-epi-PGF2α synthesis is mediated by prostaglandin H2 synthase (PGHS). To verify the role of PGHS in 8-epi-PGF2α synthesis, human PGHS-1 was expressed in COS-7 cells, a PGHS negative cell line that does not synthesize 8-epi-PGF2α. In the presence of exogenous arachidonic acid the COS-7 cells expressing human PGHS-1 produced substantial amounts of PGE2 and 8-epi-PGF2α. These data indicate that human PGHS-1 can support the synthesis of 8-epi-PGF2α and that 8-epi-PGF2α synthesis by cultured human endothelial cells during reoxygenation is dependent on the activity of PGHS-1.