It has been demonstrated that interleukin 24 (IL-24, also called melanoma differentiation associated gene 7) exerts antitumor activity. In this study, we investigated whether oncolytic adenovirus-mediated gene transfer of IL-24 could induce strong antitumor activity. A tumor-selective replicating adenovirus expressing IL-24 (ZD55-IL-24) was constructed by insertion of an IL-24 expression cassette into the ZD55 vector, which is based on deletion of the adenoviral E1B 55-kDa gene. ZD55-IL-24 could express substantially more IL-24 than Ad-IL-24 because of replication of the vector. It has been shown that ZD55-IL-24 exerted a strong cytopathic effect and significant apoptosis in tumor cells with p53 dysfunction. Moreover, no cytotoxic and apoptotic effects could be seen in normal cells infected with ZD55-IL-24. Expression of IL-24 did not interfere with viral replication induced by oncolytic adenovirus. Activation of caspase 3 and caspase 9, and induction of bax gene expression, were involved in tumor cell apoptosis induced by ZD55-IL-24. Treatment of established tumors with ZD55-IL-24 showed much stronger antitumor activity than that induced by ONYX-015 or Ad-IL- 24. These data indicated that oncolytic adenovirus expressing IL-24 could exert potential antitumor activity and offer a novel approach to cancer therapy.