The mechanism by which neutrophils [polymorphonuclear leukocyte (PMNs)] are stimulated to move across epithelial barriers at mucosal surfaces has been basically unknown in biology. IL-8 has been shown to stimulate PMNs to leave the bloodstream at a local site of mucosal inflammation, but the chemical gradient used by PMNs to move between adjacent epithelial cells and traverse the tight junction at the apical neck of these mucosal barriers has eluded identification. Our studies not only identify this factor, previously termed pathogen-elicited epithelial chemoattractant, as the eicosanoid hepoxilin A₃ (hepA₃) but also demonstrate that it is a key factor promoting the final step in PMN recruitment to sites of mucosal inflammation. We show that hepA₃ is synthesized by epithelial cells and secreted from their apical surface in response to conditions that stimulate inflammatory events. Our data further establish that hepA₃ acts to draw PMNs, via the establishment of a gradient across the epithelial tight junction complex. The functional significance of hepA₃ to target PMNs to the lumen of the gut at sites of inflammation was demonstrated by the finding that disruption of the 12-lipoxygenase pathway (required for hepA₃ production) could dramatically reduce PMN-mediated tissue trauma, demonstrating that hepA₃ is a key regulator of mucosal inflammation.