The experiments in this paper were designed to examine the therapeutic effects of adenoviral-mediated gene transfer of IFN-γ into a mouse model of an established metastatic brain tumor. Temperature-sensitive replication-defective adenovirus was generated for gene transfer of IFN-γ (AdIFN) and β-galactosidase (AdBGAL) cDNAs in vivo. In this model, treatment with AdIFN elicits prolonged survival times and brain tumor rejection. Evidence against an immune-mediated response accounting for this result include: 1) absence of a memory immune response upon challenge, 2) lack of antitumor effects at sites distal to inoculation of AdIFN, and 3) preservation of the therapeutic effects of AdIFN in scid and beige mice and in inducible NO synthase (iNOS) knockouts. High concentrations of IFN-γ do not inhibit tumor growth in vitro making it unlikely that the antitumor effect of this treatment acts directly on the growth of the tumor cells. However, gene transfer of IFN-γ inhibits neovascularization of the tumor in a 3LL-Matrigel assay in vivo, and AdIFN induces apoptosis of endothelial cells in vivo, supporting the idea that AdIFN represses tumor growth by inhibiting angiogenesis. The substantial non-immune-mediated therapeutic benefits of AdIFN in animals paves the way for devising novel strategies for treating human brain tumors.