We evaluated the antitumor effects of ionizing radiation and tumor necrosis factor-α (TNF-α) gene therapy in human malignant glioma (D54) xenografts. An adenoviral vector (Ad5) containing DNA sequences of the Egr-1 promoter was linked to a cDNA encoding the TNF-α gene (Ad. Egr-TNF). Athymic nude mice bearing D54 xenografts received intratumoral injections of Ad. Egr-TNF or the null vector (Ad. null), with and without fractionated radiation, 5 gray (Gy) per day for 6 days, a total dose of 30 Gy. Administration of Ad. Egr-TNF and 30 Gy resulted in complete tumor regression in 71% of xenografts compared with xenografts treated with radiation alone (7.4%, P= 0.006), Ad. Egr-TNF alone (0%, P= 0.012) or Ad. null with 30 Gy (0%, P= 0.002). Combined treatment with Ad. Egr-TNF and 30 Gy significantly reduced mean fractional tumor volumes compared with radiation alone (P= 0.002), Ad. Egr-TNF alone (P= 0.002) and Ad. null plus 30 Gy (P= 0.018). Histopathologic analyses of glioma xenografts treated with Ad. Egr-TNF and radiation revealed tumor vessel thrombosis by day 4 and necrosis by day 7. Thrombosis was not observed in tumors treated with Ad. Egr-TNF alone and was significantly reduced in all other treatment groups. These studies suggest that in the D54 glioma xenograft model, the antitumor effects of combining radiation and Ad. Egr-TNF are mediated, in part, by the destruction of the tumor microvasculature.