Malignant mesothelioma remains an incurable disease for which immune-modulatory therapies, such as exogenous cytokines, have shown some promise. One such cytokine, IFN-β, has potent antiproliferative and immunostimulatory activity in vitro, but its in vivo use has been limited by toxicity. We thus conducted studies evaluating intracavitary delivery of a replication-deficient adenoviral (Ad) vector encoding for the murine IFN-β gene (Ad.muIFN-β) in mouse models of malignant mesothelioma. In contrast to multiple injections of recombinant protein, a single i.p. injection of Ad.muIFN-β into animals with established tumors elicited remarkable antitumor activity leading to long-term survival in >90% of animals bearing either AB12 or AC29 i.p. mesotheliomas. A control adenovirus vector had minimal antitumor effect in vivo. Significant therapeutic effects were also seen in animals treated with large tumor burdens. Importantly, treatment of i.p. tumor also led to reduction of growth in tumors established at a distant site (flank). A number of experiments suggested that these effects were attributable to an acquired CD8⁺ T-cell-mediated response including: (a) the induction of long-lasting antitumor immunity; (b) loss of efficacy of Ad.muIFN-β in tumor-bearing, immune-deficient (SCID, SCID/beige) mice; (c) detection of high levels of specific antitumor cytolytic activity from unstimulated splenocytes harvested from Ad.muIFN-β-treated animals that was abolished by CD8⁺ T-cell depletion; and (d) abrogation of antitumor effects of Ad.muIFN-β in tumor-bearing CD8⁺ T-cell-depleted animals. These data show that intracavitary IFN-β gene therapy using an adenoviral vector provides strong CD8⁺ T-cell-mediated antitumor effects in murine models of mesothelioma and suggest that this may be a promising strategy for the treatment of localized tumors such as mesothelioma or ovarian cancer in humans.