Treatment failure after radiation therapy of prostate cancer (PC) could be a significant problem. Our objective is to design genetic radiosensitizing strategies for the treatment of PC. Cells from individuals with the genetic disorder ataxia telangiectasia (AT) are hypersensitive to ionizing radiation. Therefore, we examined whether attenuation of the AT gene product, AT mutated (ATM), in PC cells could result in an increased intrinsic radiosensitivity. A p53-mutant PC cell line, PC-3 was infected with adenoviral vectors, expressing antisense ATM RNA to various domains of the ATM gene. Immunoblot analyses of cellular extracts from antisense ATM-transfected PC-3 cells showed attenuated expression of the ATM protein within 2 days of viral infection. Compared with cells infected with an adeno-beta-galactosidase vector, antisense ATM-transfected PC-3 cells showed aberrant control of S-phase cell-cycle checkpoints after exposure to ionizing radiation. Under these conditions, the intrinsic radiosensitivity of the PC-3 cells was enhanced. Consequently antisense ATM gene therapy could serve as a paradigm for strategies that target the cellular survival mechanisms of an irradiated tumor cell and may provide therapeutic benefit to patients undergoing radiation therapy for PC.