Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-kappaB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6kappaB-EDL1E-tk, containing six copies of the NF-kappaB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6kappaB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6kappaB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6kappaB-EDL1E-tk or GCV alone. Similarly, no retardation of tumor growth was observed in mice containing LMP1-negative CT-26 tumors injected with both the p6kappaB-EDL1E-tk cassette and GCV. We propose that intratumoral injection of therapeutic agents, such as DNA of transcription-regulated cassette and GCV, via in vivo electroporation may be used as an alternative treatment for EBV LMP1-expressing cancers.