The antiviral drug ganciclovir (GCV) is toxic for mammalian cells transfected with the herpes simplex virus thymidine kinase (HSVtk) gene. To improve the results obtained by our group previously on nonviral transfection of tumor cells, we have examined here in vitro virus-free transfection of murine B16 melanoma cells via lipofectamine-nucleic acid complexes carrying either HSVtk gene transcripts or plasmid DNAs containing single and double copies of the HSVtk gene. The HSVtk gene transcripts as well as plasmids containing the HSVtk gene (s) rendered cells sensitive to GCV treatment. Tumor sensitivity to GCV conferred by the HSVtk gene transcripts was of the same level as the sensitivity conferred by plasmid DNAs containing a single copy of the HSVtk gene. However, when the plasmids containing double copies of the HSVtk gene were used, sensitivity to low GCV concentrations increased dramatically. One could appreciate this finding as an essential advantage of the plasmids containing double copies of the HSVtk gene since it allows use of the GCV concentration range which is common in clinical applications.