We examined a panel of cell lines for the expression of the myogenic proteins myoD and myogenin. High level expression of both proteins was seen in rhabdomyosarcoma (RMS). To determine whether promoter elements from these genes could direct RMS cell-specific expression, we generated reporter constructs containing one or two copies of the myoD enhancer coupled to the SV40 promoter. Transient transfection reporter assays confirmed the selective expression of beta-galactosidase (beta-gal) in 8 RMS cell lines. In contrast, very low expression from the myoD enhancer/SV40 promoter was detected in four non-RMS cell lines. To determine whether the hybrid promoter could elicit RMS-specific cytotoxicity, a mammalian expression vector containing the herpes simplex virus thymidine kinase (HSVtk) under control of the hybrid myoD enhancer/SV40 promoter was constructed. After electroporation into several cell lines, selective RMS cell kill was observed after treatment with ganciclovir. These data suggest that in vivo tumor-specific expression of HSVtk from the myoD enhancer/SV40 promoter may provide an alternative to current chemotherapy.