Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na+/K+-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1-/-), but not IFN-γ-knockout mice. Anti-CD3 mAb decreased mucosal Na+/K+-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1-/- mice. Neither α nor β subunits of Na+/K+-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3-treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na+ absorption, Na+-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR-/- mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na+/K+-ATPase activity leading to decreased intestinal Na+ and water absorption.