Abstract Prior in vivo studies have shown that indomethacin, which inhibits prostaglandin (PG) synthesis, affects fluid transport in the small bowel, enhancing spontaneous fluid absorption and reducing the amount of fluid that accumulates in response to cholera toxin and other secretory stimuli. To further explore the mechanisms involved, we determined the effects of indomethacin on ion transport, cAMP concentration, and PGE 2 production in rabbit ileal mucosa in vitro. Indomethacin (1 mM), when added alone, had no significant effect on short-circuit current (either basal or glucose-stimulated), Cl fluxes, or cAMP concentration. Indomethacin did, however, inhibit the ion transport changes caused by several secretagogues: Effects of theophylline, Ca-ionophore A23187, and arachidonate were reversibly inhibited by at least 65%, whereas effects of dibutyryl cAMP, 16,16-dimethyl PGE 2, cholera toxin, and heat-stable Escherichia coli enterotoxin were inhibited by about 30%. Indomethacin also inhibited the theophylline-evoked increase in cAMP concentration. Indomethacin decreased PGE 2 production under basal conditions and in the presence of theophylline and A23187, which may partly explain the antisecretory action of the drug. Since arachidonate increased PGE 2 release from the mucosa more than 10-fold and indomethacin did not inhibit this effect, indomethacin at high concentration (0.5–1 mM) appears to also inhibit the action of intestinal secretagogues by a prostaglandin-independent mechanism. This study also demonstrates that the antisecretory effect of indomethacin is not simply due to stimulation of an unrelated absorptive process.