Cyclophosphamide is one of the most often used anticancer drugs. Although DNA interstrand cross-links are considered responsible for its cytotoxicity, the mechanism of initiation and execution of cell death is largely unknown. Using the cyclophosphamide analogue mafosfamide, which does not need metabolic activation, we show that mafosfamide induces apoptosis dose and time dependently in lymphoblastoid cells, with clearly more apoptosis in p53(wt) cells. We identified two upstream processes that initiate apoptosis, DNA replication blockage and transcriptional inhibition. In lymphoblastoid cells, wherein DNA replication can be switched off by tetracycline, proliferation is required for inducing apoptosis at low dose mafosfamide. At high dose, transcriptional inhibition also contributes to cell death. The RNA synthesis inhibitor alpha-amanitin induced similar to mafosfamide more apoptosis in p53(wt) than in p53(mt) cells. In combination with mafosfamide, however, alpha-amanitin had no additive effect. Mafosfamide caused p53 stabilization by phosphorylation of Ser15, 20 and 37, and activation of ATM/ATR and Chk1/Chk2. Inhibition of ATM/ATR, PI3-kinase and Chk1/Chk2 by CGK733, wortmannin and DBH, respectively, attenuated the apoptotic response in p53(wt) but not p53(mt) cells. Mafosfamide induced caspase dependent apoptosis and, for low dose treated cells, caspases were preferentially activated in the S-phase, whereas at high dose caspases were activated in all cell cycle stages. These data support the conclusion that at low dose level of mafosfamide, DNA replication blockage is the dominant apoptosis-inducing event, while at high dose, transcriptional inhibition comes into play. The data provide a mechanistic explanation of why cyclophosphamide applied at therapeutic doses preferentially kills replicating tumor cells.