Platelet-activating factor (PAF) has been implicated in the pathogenesis of gastrointestinal diseases such as necrotizing enterocolitis, Crohn's disease, and ulcerative colitis. However, neither the physiologic role of PAF in the intestine, nor the mechanisms by which PAF participates in the pathogenesis of disease are well understood. The aim of the present study was to determine the direct effect of PAF on intestinal epithelial cell ion transport, and to delineate the mechanisms of regulation. Ion transport was evaluated by measuring short circuit current (Isc) in HT29-CL19A cell monolayers using Ussing chambers. PAF receptor polarity was assessed using domain-selective biotinylation followed by immunoprecipitation and streptavidin blotting of intact epithelial monolayers. PAF (1-200 μM) stimulated Isc that followed the direction of a Cl- gradient and was specifically inhibited by the Cl- channel blockers glybenclamide, 2,2′ iminodibenzoic acid and 4,4′ diisothiocyanostilbene-2, 2′ disulfonic acid, but was unaffected by the inhibition of prostaglandin synthesis with indomethacin. Stimulated Isc was only detected after apical addition of PAF, correlating with the results of biotinylation experiments indicating an exclusive apical polarity of the PAF receptor. PAF receptor antagonists CV6209 and octylonium bromide abolished PAF-stimulated Isc. Thus, mucosal acting PAF directly and specifically stimulates ion transport via activation of an apical Cl- channel in intestinal epithelial cell monolayers independent of prostaglandin biosynthesis.