In order to obtain a highly concentrated, pure and super-hot [γ-³²P] ATP, we improved the purification method of super-hot [γ-³²P] ATP which was synthesized by the method of Johnson and Walseth (1979) . The super-hot [γ-³²P] ATP was synthesized in a relatively large volume (2 ml) of reaction mixture and purified using semi-micro scale anion exchange chromatography (Dowex 1×2, 60-70μl column volume) . In combination with washing the reaction product with certain organic solvents, this chromatography technique makes it possible to obtain a highly concentrated and pure super-hot [γ-³²P] ATP (approx.7000 Ci/mmol; 20-30 mCi/ml) from [³²P] Pi of any commercial source in a good yield.