One approach to gene therapy of cancer is based on the insertion of a suicide gene into tumor cells and subsequent activation of the suicide mechanism. We used the herpes simplex virus thymidine kinase (HSVtk) gene followed by ganciclovir (GCV) treatment. The goal of our experiments was to determine the effectiveness of HSVtk gene therapy in malignant melanoma. B16BL6 murine melanoma cells retrovirally transduced with the HSVtk gene became sensitive to low concentrations of GCV. Analysis by RT-PCR showed HSVtk expression in transduced B16BL6tk+ cells. Apoptotic cell death was found in B16BL6tk+ cells treated with GCV (20 microM). The sensitivity of B16BL6tk+ cells to GCV was also examined in vivo. Tumors inoculated subcutaneously into C57BL6 mice regressed rapidly when treated with GCV (50 mg/kg twice a day) and disappeared completely after 14 days treatment. The mice remained in remission for 5 months. A bystander effect through which nontransduced B16BL6 cells were also inhibited by GCV administration when cocultured with B16BL6tk+ cells was expected. However, only slight killing of nontransduced cells was observed in vitro. Analysis of the bystander effect in vivo showed complete regression of tumors inoculated with a mixture of cells mostly consisting of B16BL6tk+ cells. A distant bystander effect was also examined. There was no regression of wild-type tumors raised at a distant site from primary B16BL6tk+ tumors. The failure of a more effective bystander effect indicates the need for further investigation of the possible use of combined gene therapy to treat melanoma.