Cancer of the rectum poses a complex therapeutic challenge because of its proximity to adjacent organs and anal sphincters. The addition of radiotherapy before surgical resection has been shown to confer good survival rates while preserving sphincter function. Nevertheless, radiation is associated with significant side effects. On the basis of our previous work showing that herpes simplex virus type-1 (HSV-1) preferentially infects human colon cancer, we set out to examine the oncolytic effect of HSV-1 on orthotopic rectal tumors in mice. Two vectors were compared for oncolytic activity, HSV-1(Gβ) with wild-type replication and an attenuated HSV-1 vector (HSV-G47Δ). Intratumoral injection of HSV-1(Gβ) and HSV-G47Δ resulted in a significant reduction or disappearance of the tumors and increased survival of mice. Although the use of HSV-1(Gβ) was associated with systemic toxicity, HSV-G47Δ appears to possess a selective oncolytic activity. Moreover, infection with HSV-G47Δ resulted in the activation of the double-stranded RNA-dependent protein kinase (PKR) pathway. A significant improvement in viral replication and the antitumor effect was observed when the PKR inhibitor 2-aminopurine was coadministered with HSV-G47Δ to the tumor. In conclusion, the efficacy of local delivery of HSV-G47Δ combined with a specific chemical inhibitor of antiviral activity points to a novel therapeutic modality for rectal cancer and other solid tumors.