The mechanism of Na(+)/H(+) exchanger 1 (NHE1) gene repression upon exposure of cells to non-apoptotic concentrations of hydrogen peroxide (H(2)O(2)) was investigated. We show that continuous presence of H(2)O(2) was not required for inhibition of NHE1 promoter activity. However, the downregulation of NHE1 promoter activity and protein expression was abrogated by the presence of beta mercaptoethanol (betaME) and dithiothreitol. The pan-caspase inhibitor zVAD-fmk also blocked the effect of H(2)O(2) on NHE1 promoter activity and expression, but unlike betaME, caspase inhibition was ineffective in rescuing the early phase of NHE1 repression. Interestingly, the effect of caspase inhibition was observed only after 9 h of exposure to H(2)O(2) and completely restored NHE1 promoter activity by 18-24 h. Using tetrapeptide inhibitors of a variety of caspases and siRNA-mediated gene silencing, caspases 3 and 6 were identified as mediators of H(2)O(2)-induced NHE1 repression, independent of initiator/amplifier caspase activation. Furthermore, incubation of cells with the iron chelator, desferioxamine, not only blocked the activities of caspases 3 and 6, but also affected NHE1 promoter and protein expression in a manner similar to zVAD-fmk. These data show that a mild oxidative stress represses NHE1 promoter activity and expression via an early oxidation phase blocked by reducing agents, and a late phase requiring an iron-dependent increase in caspases 3 and 6 activities.