Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [Isc], 76 µA · cm²; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A₂ inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1 (IL-1) but not anti-IL-1 antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01). High levels of IL-1 (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1 to the serosal side caused a potent secretory effect (Isc, 80 µA · cm²; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF- are involved in the release of the ISF. We conclude that IL-1 is probably the ISF released by macrophages in response to toxin A.