The time course of cytotoxicity induction and the bystander effect of the rabbit cytochrome P450 4B1 (cyp4B1)/4-ipomeanol (4-IM) or 2-aminoanthracene (2-AA) pharmacogene therapy systems were investigated and compared with the herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV-tk/GCV) system. Experiments were performed in rat 9L gliosarcoma cells stably expressing cyp4B1 (9L-4B1), HSV-tk (9L-tk), or their egfp (enhanced green fluorescent protein) fusion genes. Cyp4B1-mediated activation of 2-AA showed a high cell killing efficiency within only 48 hours with an onset after already 15 minutes of prodrug exposure. Residual 9L-4B1 cells were mostly damaged sublethally upon 2-AA treatment showing an S phase arrest by cell cycle analysis. 4-IM treatment of 9L-4B1 cells generated an overall weaker cell killing, especially after prodrug exposure times of less than 48 hours. Residual cells surviving 4-IM treatment showed a G2/M arrest and restarted proliferation after prodrug treatment was stopped. HSV-tk/GCV pharmacogene therapy resulted in a slower cytotoxicity induction than cyp4B1/2-AA treatment with a significantly lower cell killing efficiency after 24 and 48 hours. HSV-tk/GCV-mediated cytotoxicity was widely similar to the cytotoxicity induced by cyp4B1/4-IM with the exception of a continuous 48-hour prodrug exposure where 4-IM treatment showed a significantly higher cell killing rate. Cells surviving HSV-tk/GCV suicide gene therapy were not viable and showed an S-phase arrest. Whereas HSV-tk/GCV induced a strong bystander effect, only moderate bystander cell death depending on cell-to-cell contact was demonstrated in 9L/9L-4B1 cocultures upon 2-AA treatment and was even absent with 4-IM, thereby contrasting with earlier reports. The absence of a strong bystander effect may limit, on one hand, the overall utility of the cyp4B1 systems for cancer gene therapy. On the other hand, the weak bystander effect together with the fast induction of cytotoxicity may provide marked advantages for the use of the cyp4B1 systems as biosafety enhancers for gene marking or replacement studies and donor lymphocyte infusions after allogeneic bone marrow transplantation.