What determines the degree of cell-resistance or sensitivity to ionizing radiation is not yet known. As a corollary to the ability of ceramide to induce apoptosis, some questions arise as to whether malignant cells escape apoptosis because of their inability to mount a ceramide response to inducers of apoptosis. To shed more light on the molecular mechanisms of tumor cell response to radiation, we tested whether exposure to ionizing radiation (of 200-1000 cGy) is associated with changes in ceramide levels in A431 tumor epithelial cells and whether the ability of ceramide to induce apoptosis is inhibited by protein kinase C (PKC) activation. Our studies demonstrate an immediate decrease in cellular levels of ceramide in response to radiation, while sphingosine levels increase. Under the same conditions the cellular 1,2-diacylglycerol (DAG) levels decrease as well, being accompanied by the translocation of PKC alpha from the membrane to the cytoplasm. Elevation of membrane PKC levels by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment had no effect on cell survival after irradiation, while treatment with EGF during and after irradiation augmented cell survival. Moreover, monoclonal antibodies to the EGF receptor (EGFR) sensitize cells to radiation by facilitating radiation-induced apoptosis. It is thus plausible that in human Squamous carcinoma cells, radiation activates predominantly the EGFR to induce resistance, while both sphingomyelin and PKC signal transduction pathways are deactivated and demonstrate no significant role in the modulation of the sensitivity or the resistance of A431 cells to ionizing radiation.