In clinical gene-therapy trials for recurrent glioblastomas, transduction of the herpes simplex virus type-1 thymidine kinase (HSV-1-tk) gene with subsequent prodrug activation by ganciclovir was found to be safe, but clinical response was poor. We used positron-emission tomography (PET) with I-124-labelled 2'-fluoro-2'-deoxy-1β-D-arabino-furanosyl-5-iodo-uracil([¹²⁴I]–FIAU)—a specific marker substrate for gene expression of HSV-1-tk—to identify the location, magnitude, and extent of vector-mediated HSV-1-tk gene expression in a phase I/II clinical trial of gene therapy for recurrent glioblastoma in five patients. The extent of HSV-1-tk gene expression seemed to predict the therapeutic response. The expression of an exogenous gene introduced by gene therapy into patients with gliomas can be monitored non-invasively by PET.