The rac1 GTPase promotes oxidative stress through reactive oxygen species (ROS) production, whereas the DNA repair enzyme and transcriptional regulator redox factor-1 (ref-1) protects against cell death due to oxidative stimuli. However, the function of ref-1 in regulating intracellular oxidative stress, particularly that induced by rac1, has not been defined. We examined the role of ref-1 in vascular endothelial cell oxidative stress and apoptosis. Ref-1 was expressed in both the cytoplasm and nuclei of resting endothelial cells. Cytoplasmic ref-1 translocated to the nucleus with the oxidative trigger hypoxia/reoxygenation (H/R). Forced cytoplasmic overexpression of ref-1 suppressed H/R-induced oxidative stress (H(2)O(2) production), NF-kappaB activation, and apoptosis, and also mitigated rac1-regulated H(2)O(2) production and NF-kappaB transcriptional activity. We conclude that inhibition of oxidative stress is another mechanism by which ref-1 protects against apoptosis, and that this is achieved through modulation of cytoplasmic rac1-regulated ROS generation. This suggests a novel extra-nuclear function of ref-1.