It has been proposed that H₂O₂ increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the δ isoform of protein kinase C (PKC) was induced by H₂O₂ in CHO and COS-7 cells. H₂O₂ also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCδ, but enhanced H₂O₂-induced tyrosine phosphorylation of PKCδ in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCδ, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCδ in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.