Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.