Evidence indicates that PP2A (protein phosphatase 2A) interacts with epithelial tight junctions and negatively regulates the integrity of the tight junction. In the present study, the role of PP2A in the hydrogen peroxide-induced disruption of the tight junction was examined in Caco-2 cell monolayers. Hydrogen peroxide-induced decrease in electrical resistance and increase in inulin permeability was associated with the dephosphorylation of occludin on threonine residues. The hydrogen peroxide-induced decrease in electrical resistance, increase in inulin permeability and redistribution of occludin and ZO (zonula occludens)-1 from the intercellular junctions were significantly attenuated by selective inhibitors of PP2A (okadaic acid and fostriecin) and by knockdown of PP2A-Calpha (the catalytic subunit of PP2A). The PP2A-Calpha protein and PP2A activity were co-immunoprecipitated with occludin, and this co-immunoprecipitation was rapidly increased by hydrogen peroxide. Hydrogen peroxideinduced increase in co-immunoprecipitation of PP2A-Calpha with occludin was prevented by PP2, a Src kinase inhibitor. GST (glutathione transferase)-pull down assays using recombinant GST-Occludin-C (C-terminal tail of occludin) and the purified PP2A showed that PP2A binds to the C-terminal domain of occludin; Src-induced tyrosine phosphorylation of GST-Occludin-C enhanced this binding. The present study shows that hydrogen peroxide increases the association of PP2A with occludin by a Src kinase-dependent mechanism, and that PP2A activity is involved in hydrogen peroxide-induced disruption of tight junctions in Caco-2 cell monolayers.