Several commonly used cancer chemotherapeutic prodrugs, including cyclophosphamide and ifosfamide, are metabolized in the liver by a cytochrome P450 (CYP)-catalyzed prodrug activation reaction that is required for therapeutic activity. Preclinical studies have shown that the chemosensitivity of tumors to these prodrugs can be dramatically increased by P450 gene transfer, which confers the capability to activate the prodrug directly within the target tissue. This P450 gene-directed enzyme prodrug therapy (P450 GDEPT) greatly enhances the therapeutic effect of P450-activated anti-cancer prodrugs without increasing host toxicity associated with systemic distribution of active drug metabolites formed by the liver. The efficacy of P450 GDEPT can be enhanced by further increasing the partition ratio for tumor:liver prodrug activation in favor of increased intratumoral metabolism. This can be achieved by co-expression of P450 with the flavoenzyme NADPH-P450 reductase, which increases P450 metabolic activity, by localized prodrug delivery, or by the selective pharmacologic inhibition of liver prodrug activation. P450 GDEPT prodrug substrates are diverse in their structure, mechanism of action, and optimal prodrug-activating P450 gene; they include both established and investigational anticancer prodrugs, as well as bioreductive drugs that can be activated by P450/P450 reductase in a hypoxic tumor environment. Several strategies may be employed to achieve the tumor-selective gene delivery that is required for the success of P450 GDEPT; these include the use of tumor-targeted cellular vectors and tumor-selective oncolytic viruses. Overall, P450-based GDEPT presents several important, practical advantages over other GDEPT strategies that should facilitate the incorporation of P450 GDEPT into existing cancer treatment regimens. A recent report of clinical efficacy in a P450-based phase I/II gene therapy trial for pancreatic cancer patients supports this conclusion.