Mucosal mast cells undergo hyperplasia in a variety of inflammatory bowel diseases including nematode infection in man and animals. The intra-epithelial localization of these cells make their soluble mediators prime candidates for modulators of epithelial function. In particular previous in vivo and ex vivo studies have established a link between the release of the highly soluble mast cell granule chymases and increased mucosal permeability. The hypothesis that the rat mast cell protease, RMCP-II, directly increases permeability to macromolecules via the paracellular route is tested in this study. Monolayers of epithelial cells (Madin-Darby canine kidney cell line) were exposed to varying concentrations of RMCP-II in vitro, in the absence of other cell types or mediators, and the effect on permeability and tight junction associated proteins was investigated. Basolateral, but not apical, exposure of polarized MDCK monolayers on porous supports to RMCP-II led to concentration- (> 100 microg/ml) and time-dependent increases in electrical conductance and permeability to mannitol (MW182) and inulin (MW5000), which was accompanied by decreases in the immunostaining of the tight junction-associated proteins occludin and ZO-1. Furthermore, prolonged exposure to RMCP-II (> 12 hours) resulted in the formation of identifiable gaps separating adjacent epithelial cells, in the absence of evidence of cytotoxicity. Inhibition of RMCP-II with Soya bean trypsin inhibitor completely abrogated the response, demonstrating that proteolysis was required. These data provide direct evidence that the rat mast cell chymase RMCP-II can, in the absence of other inflammatory mediators, increase epithelial permeability via an effect on the paracellular route.