When adherent cells, such as epithelial or endothelial cells, are detached and continuously maintained in suspension, they undergo a form of programmed cell death termed anoikis. We demonstrate that coincident with endothelial cell detachment, there is a dramatic rise in the intracellular level of reactive oxygen species (ROS). Reattachment to a solid surface rapidly attenuates the level of ROS. The mitochondria appear to be the major source of the detachment-induced rise in ROS. The change in the intracellular redox state appears to contribute to endothelial anoikis, because treatment with either the cell-permeant antioxidant N-acetylcysteine or the flavin protein inhibitor diphenylene iodonium is demonstrated to reduce oxidant levels and protect against subsequent cell death. Similarly, the endogenous intracellular level of ROS is shown to correlate with the extent of cell death. Finally, we demonstrate that the activities of both caspases and of the c-Jun N-terminal kinases are modulated by the rise in intracellular ROS levels. These results suggest that oxidants serve as signaling molecules and regulators of anoikis.