Enzyme-prodrug therapy for the treatment of cancer is an experimental procedure that is under intensive investigation. However, the relative merits of the various systems for use under specific conditions are still being determined. We have compared the efficacy of cell killing by the herpesvirus thymidine kinase (HSVTK)/ganciclovir and the purine nucleoside phosphorylase (PNP)/9-(beta-M-2-deoxy-erythropentofuranosyl)6-methylpurine enzyme/prodrug systems. These were chosen because of their differential dependence on DNA replication for their mechanism of action. The HSVTK and PNP genes, expressed from the identical prostate-specific antigen promoter, were transduced into human prostate and breast cancers cells using the same human adenovirus vector. The kinetics of cell killing in the presence of the respective prodrugs was monitored using a nondestructive assay that measured total cell bioactivity. The PNP/9-(beta-D-2-deoxy-erythropentofuranosyl)6-methylpurine system was clearly superior in its ability to cause cell death in vitro. Cells were killed in about half the time and at a 5-10-fold lower input of virus relative to the HSVTK/ganciclovir system. The PNP system may offer advantages for the treatment of slow-growing tumors in which the daily proliferative rate is low or in situations in which gene delivery or expression is inefficient.