The loss of epithelial barrier function plays a crucial role in the pathogenesis of inflammatory bowel disease, with levels of 5-lipoxygenase metabolites being increased in the mucosa of these patients. The objective of this study was to determine the effect of the eicosanoids produced by the 5-lipoxygenase pathway, leukotriene B4 and D4, and 5-hydroxyeicosatetraenoic acid on epithelial barrier function. Paracellular permeability was estimated from fluorescein isothiocyanate-dextran fluxes and transepithelial electrical resistance in differentiated Caco-2 cells. Our results suggest that leukotriene D4 and 5-hydroxyeicosatetraenoic acid altered both parameters. Identification of the receptors involved in these changes indicated that cysteinyl-leukotriene receptor 1 participates in the effects of leukotriene D4. For both eicosanoids, these effects were mediated by activation of the phospholipase C/Ca(2+)/protein kinase C pathway, in addition to cAMP-independent protein kinase A activation. Furthermore, we observed a correlation between increased paracellular permeability and the redistribution of occludin, and for leukotriene D4, the disorganization of the subapical actin ring and myosin light chain kinase activation. In conclusion, on the basis of our results, we propose that 5-lipoxygenase pathway metabolites participate in the disruption of epithelial barrier function that is characteristic of inflammatory bowel disease.