The acquisition of resistance to radiation has been a matter of concern in clinical cases. However, mechanisms underlying the acquisition of radiation resistance are yet to be elucidated. We established a radiation-resistant strain (Res-HL60 cells) from HL60 leukemic cells and investigated their response to radiation. Res-HL60 cells not only proliferated on the fifth day after radiation but also had a high survival rate in a clonogenic assay. Although Chk1 was activated in HL60 cells after irradiation, the expression levels of Chk1 in Res-HL60 cells decreased. There were few differences between the two cell lines with regard to Chk2 activity. Res-HL60 cells show prolonged G2/M arrest and an early decrease in the extent of DNA damage. However, inhibitors against ATM/ATR and DNA-dependent protein kinase (DNA-PK) both abrogated the radiation resistance capacity of the cells. These results reveal that radiation-resistant strains have a high repair capacity, and inhibition of the repair system at an early stage is an effective strategy in the second round of radiation therapy.