In this study, selective expression of therapeutic transgenes was evaluated in neuroblastoma cells. Promoter fragments of the genes for neuron-specific enolase (NSEp), tyrosine hydroxylase (THp), and dopamine-beta-hydroxylase (DBHp) were studied in neuroblastoma and nonneuronal cell lines by transient transfection experiments using fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (egfp) and luciferase (luc+) assay. Both reporter gene assays revealed a neuroblastoma-selective expression mediated by NSEp and THp, whereas DBHp was active only in a murine neuroblastoma cell line. Reporter gene expression by NSEp in neuroblastoma cells was markedly higher than expression by THp, but NSEp also showed considerable background activity in nonneuronal cells. THp-driven expression of egfp was 35-fold higher in human neuroblastoma MHH-NB11 compared with nonneuronal HeLa cells. Thus, THp was chosen for a neuroblastoma-selective suicide gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-tk)/ganciclovir (GCV) system. A retrovirus vector that contained an expression cassette of a HSV-tk/egfp fusion gene and THp in antisense orientation was generated. Stably transduced human neuroblastoma cells and nonneuronal cell lines were generated, and HSV-tk/egfp expression was measured by FACS and GCV cytotoxicity assay. There was a 2.2-fold difference in green fluorescence and a 1.4-fold difference in cell killing between the human neuroblastoma MHH-NB11 and HeLa cells after HSV-tk/egfp gene transfer. The overall difference in THp-HSV-tk/egfp-mediated cell killing between neuroblastoma and nonneuronal tumor cell lines was statistically significant (P = 0.001). In conclusion, the present study demonstrated the feasibility of a neuroblastoma-selective gene therapy approach using the THp/HSV-tk/egfp expression cassette.