Gene therapy experiments in animal models have shown that apoptin expression results in tumour regression without any significant side effects. Therefore, apoptin is regarded as a potential anticancer drug for clinical applications. In this study, we analysed whether chemotherapeutic agents combined with apoptin treatment could result in enhanced cytotoxicity in human tumour cell cultures. Combined treatment with recombinant adenovirus AdAptVP3 expressing apoptin and etoposide clearly showed an additive cytotoxic effect on human osteosarcoma U2OS cells. Paclitaxel treatment combined with apoptin expression significantly inhibited the survival of p53-positive human osteosarcoma U2OS and non-small lung carcinoma A549 cells, p53-negative human osteosarcoma Saos-2 cells and p53-mutant human prostate cancer Du145 cells, already at low doses of the chemotherapeutic agent. Our results indicate that the cytotoxicity-enhancing action by the tumour-specific apoptin in combination with chemotherapeutic agents might offer an effective and safe antitumour therapeutics.