New modalities of treatment for colorectal cancer are required to support and improve those currently available. One such approach is immunotherapy by transfer of immunostimulatory genes to tumor cells. Here, we report the use of a herpes simplex virus (HSV) vector that is capable of a single round of infection (disabled infectious single-cycle [DISC]-HSV) as a gene transfer vehicle for colorectal cancer. This vector has potential advantages over other vectors for cancer immunotherapy in that it lyses infected tumor cells. Infection with DISC-HSV inhibited tumor cell growth both in vitro and in vivo. In addition, DISC-HSV-mediated cell killing occurs by both apoptotic and necrotic mechanisms. A range of colorectal tumor cell lines could be rapidly transduced with DISC-HSV/lacZ (14-90% in 4 hr). Both tumor prevention and tumor therapy protocols showed clear antitumor effects with DISC-HSV/mGM-CSF. In the prophylactic approach, an infected/irradiated whole cell vaccine protected up to 80% of mice from rechallenge. In addition, intratumoral injection of established tumors with DISC-HSV/GM-CSF caused rejection in 40% of mice and generated some protection from subsequent rechallenge. In both cases, however, it is clear that a dominant therapeutic effect of the DISC-HSV vector derives from its oncolytic properties, irrespective of the transduced gene. As a prelude to taking these studies forward to human clinical trials, we demonstrate that tumor cells could be successfully grown from freshly obtained human colorectal cancer resections (within 1 week of surgery), were transduced with DISC-HSV/hGM-CSF, and secreted the cytokine. This study provides the preclinical basis for trials of immunotherapy of colorectal cancer using DISC-HSV.