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Photodynamic Therapy (PDT) is a minimally invasive radiation therapy currently used in treatment of various cancerous and pre-malignant diseases. Photodynamic therapy with 5-aminolaevulinic acid (5-ALA/PDT) induces DNA damage following photoactivation of Protoporphyrin IX (PpIX), the photosensitizer generated from the endogenous metabolism of 5-ALA. The occurrence of DNA damage could represent one of the last causes of cell death induced by PDT. We have dissected the molecular effectors involved in the DNA damage induced by 5-ALA/PDT in several cell lines: 1. two lung adenocarcinoma cell lines, namely H1299 (p53-/-) and A549 wild type (p53+/+); 2. two sub-clones of the same cell line HCT-116, that differ for p53 expression (p53+/+ and p53-/-) and 3. a prostate adenocarcinoma cell line, PC3 (p53-/-). We have focused our attention on p53, which controls the DNA damage response and on the efflux regulator ATP binding cassette transporter G2 (ABCG2). The levels and the activity of these two gene products are relevant for the survival in cells exposed to photodynamic treatment. Cell cycle, DNA damage (phosphorylated γ-H2AX and Comet assay) and ABCG2 levels were measured before or after irradiation. We found that all cell lines, except A549 and PC3, underwent extensive DNA damage upon PDT. The resistance of A549 and PC3 cells to photodynamic DNA damage was due to the high levels of ABCG2 expression upon irradiation. In fact, these cells displayed high levels of DNA damage signatures and underwent death when the ABCG2 was inhibited. Analysis of ABCG2 expression shows a complex regulation at transcriptional and post-transcriptional levels and its expression was dependent on oxidative stress. On the other hand, p53 expression or activity did not exert a significant effect on 5-ALA-induced cell death. Taken together, all these findings provide novel information on the role of the ABCG2 transporter in oxidative stress and a new tool to manipulate resistance to photodynamic therapy.