Given previous work showing that an adenoviral vector expressing IFN-beta (Ad.IFNbeta) was highly effective in eradicating i.p. mesothelioma tumors, the antitumor efficacy of this agent was evaluated in an orthotopic model of bronchogenic adenocarcinoma of the lung. These transgenic mice have a conditionally expressed, oncogenic K-rasG12D allele that can be activated by intratracheal administration of an adenovirus expressing Cre recombinase (Ad.Cre). K-rasG12D mutant mice were given Ad.Cre intranasally to activate the oncogene. Mice were then given 10(9) plaque-forming units of a control vector (Ad.LacZ) or Ad.IFNbeta intranasally 3 and 4 weeks later, a time when lung tumors had been established. Cells derived from K-ras-mutated lung tumors were also grown in the flanks of mice to study mechanisms of therapeutic responses. In two separate experiments, untreated tumor-bearing mice all died by day 57 (median survival, 49 days). Ad.LacZ-treated mice all died by day 71 (median survival, 65 days). In contrast, 90% to 100% of mice treated with Ad.IFNbeta were long-term survivors (>120 days; P < 0.001). In addition, immunity to re-challenge with tumor cells was induced. In vitro and flank tumor studies showed that Ad.IFNbeta induced direct tumor cell killing and that depleting natural killer or CD8+ T cells, but not CD4+ T cells, with antibodies attenuated the effect of Ad.IFNbeta. These studies, showing remarkable antitumor activity in this orthotopic lung cancer model, provide strong preclinical support for a trial of Ad.IFNbeta to treat human non-small cell lung cancer.