Protein phosphorylation and dephosphorylation were key regulatory mechanisms in signal transduction as well as in catabolisim of carbohydrates, photosynthesis, the growth of the cell and expression of the gene, etc. Therefore, to establish a sound method of protein phosphorylation assay is of much importance. Ataxia-telangiectasia mutated(ATM) was the product of the gene mutated in the human genetic disorder ataxia-telangeictasia(AT),which regulated the cell's biology response of DNA damage by phosphorylating a series of proteins involved in cell cycle checkpoints, DNA repair and apoptosis. It seemed that ATM played a central role in radiation-induced activation of the tumour suppressor gene product p53. The recombinant protein GST-p53 was expressed in E.coli and purifed by affinity chromatography. Then, ATM was immunoprecipitated from HeLa cells exposed to 10 Gy γ-ray; and the purified GST-p53 was incubated with immunoprecipitated ATM and ［γ-32P］ATP. It turned out that the immunoprecipitated complex of ATM could phosphorylate GST-p53 in vitro. Moreover, the standardization of this method facilitated delineating protein phosphorylation by protein kinase and screening substitutes of other protein kinases.