The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H₂O₂ was determined in Caco-2 cell monolayer. Incubation with H₂O₂ (20μM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170–250kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H₂O₂. However, H₂O₂ reduced 80–85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H₂O₂ reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H₂O₂-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H₂O₂ inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.