Cultured intestinal epithelial monolayers serve as models for mechanistic studies of intestinal inflammation. One crucial aspect of epithelial function modulated by inflammation is permeability. Indices of permeability typically obtained are transepithelial resistance or more formalized assays using Ussing chambers modified for cultured monolayers. Such Ussing chamber systems are inconvenient for screening assays and their geometry precludes evaluation of important inflammatory responses such as transepithelial migration of neutrophils. Here we use a novel flux assay to investigate the kinetics of fluorescein-labelled dextran (FD) molecules across intestinal epithelial cell lines grown on permeable membrane supports. Our results show that we could consistently demonstrate picomolar flux of FD in a range of sizes (3-70 kD) across T84 epithelial monolayers. Such FD flux was time- and dose-dependent and flux increased exponentially with decreasing transepithelial resistance. Exposure of intestinal epithelia to mucosal-derived lymphocytes or to soluble lymphocyte products (interferon-gamma, interleukin-4 or interleukin-13) increased FD flux in a dose-dependent fashion. Finally, studies of neutrophil transepithelial migration revealed qualitative and quantitative differences in FD flux depending on FD size. We conclude that in vitro transepithelial FD flux may be a useful tool to study aspects of intestinal permeability in health and in disease.