The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP(4) receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP(4) receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP(4) did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR(inh)172. Co-expression of CFTR and EP(4) resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR(inh)172. The EC(50) for lubiprostone mediated CFTR activation was ~10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP(4) receptor in oocytes.