Epithelial barrier dysfunction contributes to progression of intestinal and systemic disease. However, there is a fundamental gap that separates clinicopathologic significance from molecular understanding of the mechanisms responsible for barrier regulation. The major components of the tight junction, which forms the paracellular barrier, have been identified over the past two decades and fall into three major groups; scaffold proteins, e.g. ZO-1; transmembrane regulatory proteins, e.g. occludin; and pore-forming proteins, e.g. claudins. How these proteins interact to regulate the barrier is incompletely understood. Thus, tight junction biology is at a crossroads that requires a transition from protein discovery to identification of the means by which these proteins interact to regulate function. To better understand these interactions, we have recently applied in vitro and in vivo fluorescence recovery after photobleaching (FRAP) analyses with in vitro protein binding studies and measures of tight junction barrier function. The data indicate that modulation of tight junction protein intermolecular interactions is a mechanism of barrier regulation, and implicate ZO-1 as a critical intermediate in these molecular events. Moreover, disruption of these regulatory processes can reverse cytokine-induced barrier dysfunction. Thus, in addition to providing novel insight into the mechanisms of physiological and pathophysiological barrier regulation, these data provide new opportunities for therapeutic intervention.