We studied the genotoxic and apoptosis-inducing properties of ganciclovir (GCV) and penciclovir (PCV) using Chinese hamster ovary cells stably transfected with the thymidine kinase (tk) gene of herpes simplex virus-1 (HSV-1). Cells expressing HSVtk were 300 and 100 times more sensitive than their isogenic HSVtk- counterparts to the cytotoxic effects of GCV and PCV, respectively. Using radiolabeled drugs, GCV was found to be incorporated into the genomic DNA much more effectively than PCV. GCV was highly potent in inducing chromosomal aberrations compared with PCV, which provoked less sister chromatid exchanges and chromosomal changes using equimolar or equitoxic doses. For both agents, apoptosis was shown to be the major route of cell killing. Time course experiments revealed that neither genotoxicity nor apoptosis were induced within the cell cycle exposed to the drug; they are late events provoked in the following cell cycle(s). This indicates that the incorporation/exposure step of GCV or PCV into DNA is not decisive for triggering genotoxicity and apoptosis, but that events occurring subsequently, presumably during replication of a DNA containing the nucleotide analogs, are of major importance. Because PCV, unlike GCV, induced highly effectively apoptosis without exerting much genotoxicity, the use of PCV as a relatively safe alternative drug for suicide gene therapy of malignant diseases is recommended.