Hydrogen peroxide (H₂O₂) is a reactive oxygen species that can be produced in the digestive tract by inflammatory cells or during reperfusion following ischemia. To evaluate a possible direct effect of H₂O₂ on epithelial secretory cells, well-differentiated colonie T₈₄ cells were grown to confluence on permeable membranes and studied in Ussing chambers. In this model, where the measured short-circuit current (Isc) reflects electrogenic secretion, we observed that H₂O₂ stimulated a concentration-dependent and transient secretory response: 5.5 mM H₂O₂ produced a peak Isc of 12.4 μA/cm² after 4 min, 2.2 mM H₂O₂ a peak Isc of 7.9 μA/cm² after 4 min, and 1.1 mM H₂O₂ a peak Isc of 5.5 μA/cm² after 16 min (N = 5). When 97 experiments using 5.5 mM H₂O₂ were reviewed, the mean peak Isc response was 8.9 ± 0.5 μA/cm². A similar secretory response was elicited whether H₂O₂; was added to the serosal, to the mucosal, or simultaneously to both sides of the T₈₄ cell monolayer. This secretory response reflected transcellular chloride secretion because it was inhibited by the depletion of chloride in the medium and by the suppression of the Na⁺ K⁺,2C1 co-transporter activity necessary for the chloride gradient driving chloride secretion. When T₈₄ cell monolayer resistance was studied, 5.5 mM H₂O₂ produced a transient decrease in resistance, reflecting transcellular chloride secretion, and a gradual decline in resistance (75% of the initial value after 55 min). The secretory response to H₂O₂ was increased 2-fold in T₈₄ cells maximally stimulated with 10 nM vasoactive intestinal peptide (VIP), a neuropeptide which acts via cAMP, demonstrating synergism between the two agents. In contrast, the secretory responses produced by H₂O₂ and carbachol, which acts through the Ca SU2+ pathway, were additive. A late inhibitory effect of H₂O₂ was also observed: in cells previously treated with 5.5 mM H₂O₂, the subsequent secretory responses to either VIP or carbachol were partially inhibited. These secretory effects were specific for the oxidant properties of H₂O₂ because they were inhibited by 450 U/ML catalase and by 5 mM dithiothreitol, but were unaffected by 50 μM deferoxamine B or Fe³⁺. H₂O₂ may be a potential modulator of intestinal or colonic secretion in certain pathologic conditions such as inflammation or ischemiareperfusion.