Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.