Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E₂ (PGE₂) stimulates CFTR-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominately achieve their effects via binding to prostanoid receptors, we hypothesised that this 8-iso-PGE₂ stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence we here demonstrate that Calu-3 cells express the EP_1-4 and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl^- efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE₂, misoprostol (EP₂, EP₃ and EP₄) and PGE₁-OH (EP₃ and EP₄) stimulate iodide efflux; however, iloprost, butaprost, sulprostone and fluoprostenol (agonists of the EP₁, EP₂, EP₃ and FP receptors respectively) have no effect. The iodide efflux seen with 8-iso-PGE₂ is abolished by the EP₄ receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP₄ subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP₄ receptor. We propose that 8-iso-PGE₂, acting via EP₄ receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.