Gene directed-enzyme prodrug therapy (GDEPT) is an approach for local sensitization of target tumor cells to an enzymatically activated, otherwise non-toxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector has the original murine leukemia virus (MLV)-derived U3 region of the 3¿LTR replaced with the mouse mammary tumor virus (MMTV) promoter known to be specifically active in the mammary glands of transgenic animals. Upon infection of target cells and in the course of reverse transcription the resulting rearrangement of the vector causes the expression of the therapeutic CYP2B1 gene to be controlled by the MMTV promoter. The vector was designed for future use with encapsulated virus producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself that can deliver the CYP2B1 gene to the surrounding tumor cells. The generated vector was shown to be effectively packaged in virus producing cells and, subsequently, to facilitate production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that upon infection the effective IFA dose was reduced by sixteen fold. Infection efficiencies in vivo were determined using a reporter gene bearing vector virus in a mammary cancer cell-derived xenograft tumor mouse model.