Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of “marker gene” and “marker substrate” in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK⁺ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2′-fluoro-2′deoxy-1-β-d-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2′-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK⁺ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[¹⁴C]FIAU accumulation was shown to be high in RG2TK⁺ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumuiated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.