TRAIL is a member of the tumor necrosis factor superfamily that induces apoptosis in a variety of tumor cell types both in vitro and in vivo, while demonstrating minimal cytotoxicity toward normal tissues. One disadvantage to previous in vivo protocols was the need for large quantities of TRAIL to suppress tumor growth. Here we engineered a replication-deficient adenovirus to encode human TNFSF10 (Ad5-TRAIL) as an alternative to recombinant, soluble TRAIL protein. The results show that TRAIL-sensitive prostate tumor cell targets infected with Ad5-TRAIL undergo apoptosis through the production and expression of TRAIL protein. This activity was limited to TRAIL-sensitive tumor cells, as normal prostate epithelial cells were not killed by Ad5-TRAIL. Furthermore, in vivo administration of Ad5-TRAIL at the site of tumor implantation suppressed the outgrowth of human prostate tumor xenografts in SCID mice. Histologic examination of prostate tumors treated locally with Ad5-TRAIL revealed areas of apoptosis within 24 hours of injection. These results further define Ad5-TRAIL as a novel anti-tumor therapeutic and demonstrate its potential use as a means for treating prostate tumors, as well as other solid tumors, in vivo.