ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3-kinase (PI3-kinase). In order to determine the contribution of ATM and DNA-PKcs to the increased sensitivity of cells to DNA-damaging agents observed in the presence of PI3-kinase inhibitors, we examined the effects of a PI3-kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X-irradiation and ultraviolet (UV)-irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X-irradiation, but not to MMC or UV-irradiation. Hypersensitivity to BLM and X-irradiation and normal sensitivity to MMC and UV-irradiation are characteristic phenotypes of both AT and scid mice. DNA-dependent protein kinase (DNA-PK) activity was suppressed by wortmannin to 45–65% of the control values in all of the cells except SCF, in which DNA-PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X-irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA-PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy. Int. J. Cancer 78:642–647, 1998. © 1998 Wiley-Liss, Inc.