We found that hydrogen peroxide (H2O2) enhances EGF receptor tyrosine phosphorylation in intact cells as well as solubilized membrane of an EGF receptor hyperproducing cell line NA. An antioxidant MnCl2 effectively inhibited this enhancement. Interestingly, overall phosphorylation of the EGF receptor enhanced by H2O2 was half that of the EGF-enhanced phosphorylation when the receptor immunoprecipitated from [32P]orthophosphate-labeled cells was examined. Tryptic phospho-peptide mapping of these receptors revealed that EGF enhanced the phosphorylation on five specific residues including serine 671, 1,046 and 1,047, threonine 669 and tyrosine 1,173, whereas H2O2 enhanced the phosphorylation remarkably on tyrosine 1,173 and three other residues and only moderately on serine 1,046 and 1,047 and threonine 669. Thus, H2O2 preferentially enhances the tyrosine phosphorylation of EGF receptor through oxidant stress.