The tight junction seals the paracellular space between adjacent epithelial cells. However, the seal is neither absolute nor fixed, and both size- and charge-selectivity of the barrier can be modulated. We and others have shown that myosin light chain kinase (MLCK) is a critical mediator of tight junction barrier regulation, particularly with regard to size-selectivity, as part of physiological and pathophysiological processes (1-4). However, the intracellular effectors of MLCK-dependent barrier regulation have not been defined and data describing the impact of such regulation in vivo are limited. We have recently reported that the tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state (5). We hypothesized that this remodeling might contribute to barrier function and, therefore, that alterations in tight junction protein exchange behavior could be related to barrier regulation. Here, we show that two tight junction proteins, ZO-1 and occludin, play specific roles in MLCK-dependent barrier regulation following exposure to physiological and pathophysiological stimuli. Analysis of fluorescence recovery after photobleaching (FRAP), showed that the increased barrier function that followed MLCK inhibition stabilized ZO-1 at the tight junction, but did not affect claudin-1, occludin, or actin exchange. ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition and free ABR acted as a dominant negative to prevent barrier function increases following MLCK inhibition, and MLCK inhibition did not regulate the barrier in cells lacking ZO-1. Thus, the ZO-1 ABR and ongoing ZO-1 exchange are essential to MLCK-dependent barrier regulation. To better understand the consequences of MLCK-dependent barrier regulation in vivo, we developed transgenic mice expressing constitutively-active MLCK (CA-MLCK) within intestinal epithelia (6). These mice demonstrated increased paracellular flux of both macromolecular solutes and cations. However, CA-MLCK expression only increased macromolecular paracellular flux in vitro. Further analysis showed that mucosal IL-13 expression is increased by intestinal epithelial CA-MLCK expression in vivo and that this triggers claudin-2 synthesis and increased paracellular cation flux. Thus, MLCK-dependent permeability increases macromolecular paracellular flux to induce mucosal immune activation that, in turn, feeds back on the tight junction to regulate paracellular cation permeability. We speculate that interactions between these otherwise distinct tight junction paracellular pathways may contribute to disease pathogenesis.