Studies of tumor cell lines expressing individual cytochrome P450 genes are essential for evaluation of the utility of P450 prodrug activation-based cancer gene therapy (1). P450-expressing tumor cells may also be useful to identify novel P450 gene /prodrug combinations (see Chapter 5). The evaluation of candidate P450 genes for use in prodrug activation gene therapy is greatly facilitated by the availability of P450-expressing tumor cell lines, which can be prepared by the retroviral transduction methods described in this chapter. Finally, it should be noted that the methods described in this chapter are not limited to P450 gene transduction, and can readily be applied to the transduction of other genes of interest to cancer gene therapy. In vitro cytotoxicity assays using these cell lines can then be carried out as described in Chapter 7 prior to initiating more costly and labor intensive in vivo tumor studies in animal models (described in Chapter 8).