Human papillomavirus type 16 (HPV-16) is associated with development of anogenital squamous cell cancers (SCCs) and their precursor, intraepithelial neoplasia (IN). Few approaches to the treatment of IN to prevent SCC are targeted specifically to HPV. We have designed an HPV-specific therapy using the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene driven by an HPV-specific promoter in the HPV-16 long control region (LCR) (nucleotide 7450-nucleotide 104), which is regulated by the HPV E2 protein. Expression of the HSV-1 TK gene is designed to render HPV-infected cells sensitive to the prodrugs ganciclovir (GCV) and acyclovir (ACV). To assess the E2 specificity of gene expression driven by the HPV-16 LCR, we measured luciferase expression in HPV-positive and HPV-negative cell lines. Significant induction of luciferase activity was observed in HPV-positive cells when compared with four different HPV-negative epithelial cell lines. Cotransfection of an HPV-negative cell line, MDCK, with an HPV-16 E2-expressing plasmid resulted in 15- to 20-fold induction of luciferase activity, suggesting specific activation by E2 protein. A plasmid expressing the HSV-1 TK gene driven by the LCR was transfected into CaSki and SiHa cells. Treatment of transfected cells with either GCV or ACV (20-30 microg/ml) for 6-10 days resulted in 80-95% cell death. Cell death was progressive, dose dependent, and mediated by apoptosis. These results suggest that direct gene transfer of the HSV-1 TK gene into HPV-16-infected cells expressing E2 protein, accompanied by treatment with either GCV or ACV, may be a clinically feasible therapeutic strategy.