Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID‐associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco‐2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca⁺⁺ modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter‐grown Caco‐2 intestinal monolayers in Ca⁺⁺‐free solution (CFS), consisting of modified Krebs‐buffer solution containing 0 mM Ca⁺⁺ and 1 mM EGTA, resulted in a rapid drop in Caco‐2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco‐2 TJ permeability was rapidly reversed by the re‐introduction of Ca⁺⁺ (1.8 mM) into the incubation medium. The CFS‐induced increase in Caco‐2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO‐1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS‐induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri‐junctional actin and myosin filaments. The inhibition of CFS‐induced activation of Caco‐2 MLCK with MLCK inhibitor (ML‐7) prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS‐induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco‐2 MLCK activation and the resultant contraction of the peri‐junctionally located actin‐myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg⁺⁺‐myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca⁺⁺ is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca⁺⁺ from the incubation medium causes activation of Caco‐2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability. Microsc. Res. Tech. 51:156–168, 2000. © 2000 Wiley‐Liss, Inc.