Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-α (PFT-α), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and -proficient glioma. We observed significant interaction of PFT-α with radiation-induced G(1) checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-α-mediated inhibition of radiation-induced expression of the p53 target gene p21(Waf1). Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-α in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-α significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-α treatment. In conclusion, PFT-α might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and -deficient tumors.