The thymidine kinase gene (tk) of herpes simplex type 1 virus (HSV-1) was inserted into a retroviral vector under the transcriptional control of the enhancer-promoter element of the Moloney murine leukemia virus long terminal repeat. Replication-defective viral particles were obtained by transfection of vector DNA into the packaging cell line psi2 and were used to infect C6 rat glioma-derived cell lines in culture. The sensitivity of these cells to the toxic effects of the nucleoside analog ganciclovir was found to be significantly increased by transfer of the HSV-1 tk gene. The difference in sensitivity between infected and uninfected cells defined ganciclovir concentrations that could be used to selectively kill essentially all infected cells while sparing uninfected ones. C6 glioma cells introduced subcutaneously into nude mice were highly tumorigenic. Growth of tumors produced from C6-derived cells bearing the HSV-1 tk gene, but not parental C6 cells, could be inhibited by intraperitoneal administration of ganciclovir. This work demonstrates the effectiveness of the thymidine kinase expressed by the HSV-1 ks gene in sensitizing brain tumor cells to the toxic effects of nucleoside analogs. Retrovirus vectors should thus prove useful in the selective delivery of this killer gene to dividing tumor cells in the nervous system, where most endogenous cells are not dividing.