In the present study we characterized the functional and structural disruption of the paracellular barrier of intestinal epithelium in vitro in relation to cytotoxicity after apical Cd2+ exposure. For that purpose filter-grown Caco-2 and IEC-18 cells were apically exposed to 5 to 100 μM CdCl2 for 4 or 14 h. It was found that the effects of Cd2+ on the epithelial barrier were concentration- and time-dependent. The first detected effects of Cd2+ in Caco-2 cells after 4 h exposure were a decrease in transepithelial electrical resistance, increased permeabilities of mannitol and PEG-4000, and changes in intercellular localization of ZO-1, occludin, and e-cadherin. The effects were far more pronounced after prolonged exposure. The disruption of the paracellular barrier by 5 to 30 μM Cd2+ was detected without a significant loss of viability of the Caco-2 cells. In the IEC-18 cells, Cd2+ concentrations affecting the barrier (50 and 100 μM) also affected cell viability. In both cell lines the effects on the cell layers continued to develop after removal of extracellular Cd2+. This correlated with the cellular retention of Cd2+, which was high for the 12 h following 4 h accumulation. This study showed that the decreased epithelial barrier function of intestinal epithelial cells is accompanied by tight junction disruption. It is concluded that Cd2+ causes increased paracellular permeability by disruption of junctional function and structure. The initial junctional effects of Cd2+ suggest that Cd2+ increases its own bioavailability by causing disruption of the intestinal paracellular barrier.