BACKGROUND Mild psoralen and UV (PUV) treatments inactivate viral DNA replication, but the virus retains its ability to infect cells. Thus, PUV treatment of vaccinia virus (VV) vectors may increase the safety of gene delivery and extend the duration of gene expression. Although the first studies on PUV-inactivated VV (PUV-VV) for the delivery of suicide or cytokine genes to cancer cells were promising, the efficiency and kinetics of exogenous gene expression have not been fully evaluated. Furthermore, these studies should be extended to other gene therapy strategies, e.g. tumor suppressor genes. METHODS We constructed VV recombinants carrying the luciferase (luc) gene, or the tumor suppressor p53 gene, to analyze exogenous gene expression after PUV treatment. Apoptosis induction and antitumor effects were examined in glioma cell culture and in an animal model, respectively. RESULTS PUV-VV induced efficient PE/L-driven expression of luc and p53 exogenous genes in infected cells. A surprising prolonged p53 protein production was measured in glioma cells infected with PUV-VV expressing p53 (VV-TK-53) on Days 5-7 post-infection, reaching a maximal level of 9 microg/ml. VV-TK-53 induced apoptosis in 88% and 77.6% of infected C6 and 9L glioma cells, respectively. In contrast, 80% of cells infected with the PUV-inactivated control virus remained viable. Finally, ex vivo infection of C6 glioma cells with PUV-inactivated VV-TK-53 significantly reduced subsequent tumor growth in nude mice. CONCLUSIONS Replication-deficient PUV-VV is safe and very efficient in prolonged foreign gene expression. Therefore PUV-VVs are recommended as vectors for applications in cancer gene therapy and recombinant vaccine development.