Abstract: Objective To evaluate human prostate carcinoma cells as targets for herpes simplex virus thymidine (HSV-TK)-mediated gene therapy, we tested the utility of adenoviral vectors on three human cell lines LNCaP,PC-3,and MRC-5.Our viral vectors carried a fusion gene of HSV-TK and an enchanced florescent protein for accurate determination of the gene transfer rate and its contribution to the treatment results in each case. Methods We use a recombinant adenovirus vector containing the enhanced green fluorescent protein （EGFP）gene. Cells transfected with recombinant adenovirus expressed green fluorescent protein. The percent of transfection was determined by flurorescence microscopy. To assay the cytolytic effects of Ad-hTERT-HSV/TK on tumor and normal cells, we performed a Cytopathic Effect test. MTT assay was used to determine cell viability at various GCV concentrations. DNA ladder assay was carried out to show the apoptosis of transferred cells. Results Ad-hTERT-EGFP was observed to transfer efficiently in prostate cancer cells but poorly in normal cells under fluorescence microscope. Cytopathic Effect test and MTT assay showed that Ad-hTERT-HSV/TK selectively transferred and killed tumor cells while leaving normal cells unaffected. Conclusion Recombinant adenovirus vector containing EGFP can report percent of transfection correctly and conveniently. The human prostate cancer cells LNCaP is more sensitive to the treatment of AdTK/GCV system than PC-3. In summary, we demonstrate a new strategy for prostate cancer biotherapy and it has potential application in clinic.