We have examined the effect of abrogation of the G₂ checkpoint on the radiosensitivity of G₁ checkpoint-proficient and G₁ checkpoint-deficient cells. A549 human lung adenocarcinoma cells were transduced with the E6 oncogene of the human papillomavirus type 16 to eliminate their radiation-induced G₁ arrest. These E6+ cells exhibited a dose-dependent increase in radiation resistance compared to control A549 cells transduced with the vector alone. Treatment (96 h) with 2 mm caffeine resulted in an abrogation of the cellular G₂ checkpoint in both E6+ and control cells and a differential radiosensitizing effect on the two cell lines such that the E6+ clones and the vector controls became equally radiosensitive. These data show that human tumors which are radioresistant due to the loss of the p53-mediated G₁ checkpoint can be made radiosensitive by abrogation of the G₂ checkpoint. The implications of these results for cancer therapy are discussed.