In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.