Adeno-associated viral (AAV) vectors preferentially integrate into the genome of cells that are defective in DNA repair, such as occurs with DNA-PKcs deficiency or poly(ADP-ribose) polymerase-1 down-regulation. As the tumor suppressor protein p53 regulates the transcription of many genes involved in DNA repair, we sought to determine whether functional p53 affects the efficiency of AAV integration. p53 is mutated in more than 50% of cancers, and site-specific integration of AAV into the AAVS1 site of human chromosome 19 has frequently been observed in transformed cancer cell lines, but rarely seen in primary cells or in vivo. We therefore hypothesized that p53-negative cells would be more permissive to AAV integration than p53-positive cells. The integration of a rep-and green fluorescent protein-encoding recombinant AAV vector was quantified in p53-expressing and p53-deficient HCT116 colon cancer cells. Our results show that there is a higher efficiency of AAV integration in p53-negative cells compared with p53-positive cells, indicating that p53 does indeed inhibit AAV integration. Further experiments suggest that this p53-mediated block to AAV integration is likely to be through binding of p53 to the AAV Rep protein and the consequent inhibition of Rep activity during AAV integration.