An emerging strategy for cancer gene therapy involves the transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells, rendering them susceptible to the cytotoxic effects of ganciclovir. The observation that HSV-tk-expressing cells can also induce cell death in neighboring cells, which do not express HSV-tk, has been called the bystander effect. Gap junction-mediated transfer of cytotoxic molecules to bystander cells may be an important mechanism of bystander cell death, although others have suggested a role for phagocytosis. In this study, we evaluated the mode of cell death in bystander cells. We detected apoptosis in bystander cells and found that bystander cell death could be inhibited by BCL2 expression. We determined that ganciclovir incubations for 10 h were sufficient to induce cell death in most bystander cells cocultured with HSV-tk-expressing cells. During this period, no phagocytosis was detected, although it was obvious at later stages.