Tumor cells transduced with a retroviral vector expressing a herpes virus thymidine kinase (HSV tk) gene are rendered sensitive to the antiherpetic drug, ganciclovir. The bystander effect refers to the observation that not all cells need be transduced to eradicate the cell population by treatment with ganciclovir. We demonstrate that metabolic cooperation can account for this bystander effect. When HT1080 human fibrosarcoma cells marked with a lacZ gene (LZ+5) were cocultured with HT1080 cells transduced with a retrovirus expressing HSVtk (HT1080tk11), at a density at which the majority of cells were in contact, both HT1080tk11 and LZ+5 cells were killed by ganciclovir. When cells were cocultured at a low density where the majority of cells are not in contact with one another, however, only the HT1080tk11 cells were killed. This result suggests that cell contact with HT1080tk11 cells is necessary to render the HSVtk- LZ+5 cells sensitive to ganciclovir. Because involvement of metabolic cooperation in the killing of the LZ+5 cells would require not only contact between HT1080tk11 and LZ+5 cells but also the capacity to transfer small cytotoxic molecules from the former cell to the latter, transfer of radioactive molecules between the two cell lines was assessed by autoradiography after treatment of a coculture with [3H]ganciclovir. Isolated HT1080tk11 cells incorporated the labeled ganciclovir into their nuclei, whereas isolated LZ+5 cells did not. LZ+5 cells incorporated [3H]ganciclovir, only when in contact with HT1080tk11 cells. These findings indicate that a ganciclovir metabolic product, presumably a phosphorylated form, can pass from HSV tk+ to HSV tk- cells and mediate cytotoxicity as a consequence of direct contact.