E. coli (STa) enterotoxin reduces net fluid absorption but stimulation by STa of epithelial cell chloride ion and resultant fluid secretion could also contribute to diarrhoeal disease. Yet, contrary to expectations, net secretion does not occur in vivo when perfusate recovery methods are used (1) although inhibition of absorption does. Experiments were done with zero sodium ion perfusates and hence minimal fluid uptake, in the expectation that STa-stimulated secretion might then become evident. Fluid uptake from recirculated loops was measured in anaesthetised (70 mg/kg i.p. Sagatal) unfasted Sprague-Dawley rats using recovered perfusate to assess fluid absorption. Twenty five cm loops of proximal jejunum were perfused with buffers in which either choline or mannitol replaced sodium ion. At the end of the experiment, the animals were humanely killed. In zero sodium ion perfusates containing choline ion, net fluid absorption was negative 12.1 ± 3.3(9) μl/cm/h, further reduced (p<0.05) to negative 29.2 ± 4.2(8) μl/cm/h by STa. As previously (1), to determine whether the small effect of STa in the nominal absence of sodium ion depended on persisting sodium ion uptake, 0.1 mM of the NHE-3 inhibitor EIPA (ethyl-iso-propyl-amiloride) was added. In the zero sodium ion perfusates with EIPA, fluid movement of –10.8 ± 3.8(8) μl/cm/h did not differ from –14.6 ± 6.2(6) μl/cm/h with STa. These experiments indicate that when fluid uptake is inhibited by inhibiting sodium ion uptake, there is no subsequently detectable effect of STa on fluid movement, attributable to secretion. Similar results were obtained when mannitol substitution was used. Similarly, extending previous work with bumetanide (2), the chloride channel blocker NPPB did not restore STa-inhibited fluid absorption since fluid transport was 22.3 ± 4.2(6) μl/cm/h after STa exposure and 34.1 ± 8.0(6) μl/cm/h with STa + NPPB, although both were lower than control values of 80.0 ± 14.0(6) μl/cm/h. These observations further challenge the concept of chloride-ion-dependent epithelial fluid secretion as a basis for enterotoxin action.