Gene transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene sensitizes tumor cells to the toxic effect of ganciclovir (GCV). The toxic effect of GCV extends to nontransduced surrounding cells by a metabolic process known as the bystander effect. A distant bystander effect, which involves anatomically separated tumors, has been reported in vivo. Our aim was to evaluate and characterize such distant effect in a rat model of colorectal tumors implanted in the liver using adenovirus to carry the HSV-tk gene. Two colorectal tumors were implanted in two distinct liver lobes of the liver. One of the tumor was transduced with an adenoviral vector containing HSV-tk gene. The volumes of the tumors were monitored after GCV treatment. Implication of the immune system was studied histologically and after in vivo manipulations. After GCV administration, the nontransduced distant tumor regressed partially or completely in the experimental group. Immunohistochemical analysis revealed the presence of CD8+ lymphocytes in the distant lesion. HSV-tk/GCV-induced immune response against tumors was evidenced by an adoptive transfer assay (Winn assay) and the distant bystander effect was blunted after CD8+ lymphocytes depletion. However, the survival rates for treated animals were not improved. These findings demonstrate that an immune-mediated effective distant bystander effect can be obtained after limited adenoviral-mediated transfer of the HSV-tk gene.