Abstract: Objective:To investigate the target killing effect of hypoxia reaction element promoter-thymidine kinase/ganciclovior (HRE-TIC/GCV) system on human osteosarcoma cells under hypoxia conditions. Methods: Recombinant eukaryotic expression plasmid pBI-HRE-HyTK was constructed containing Hygromycin phosphotransferase-thymidine kinase, (HyTK)fusion gene and HRE promoter. The recombinant vectors were transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. The methods of PCR and RT-PCR were used to confirm existence and expres-sign of TIC gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HRE-TIC/ GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed coculture experiment. The mechanism of cytotoxicity on cells was analyzed by flow cytometry and electron micro-scope. Results: The eukaryotic expression plasmid pBI-HRE-HyTK was constructed successfully. The transfected cell line MG63 with was harvested, and DNA existence and mRNA expression of TIC gene was demonstrated. Under normoxia conditions only 30% MG63TK cells were killed when exposure to 50ug/ml of GCV; However 50% cells were killed with 1 N., g/ml of GCV and more than 90% cells were killed with 50ug/ml of GCV under hypoxia conditions. When MG63 and MG63TK cells were cocultured, the BSE of MG63TK increased distinctly under hypoxia conditions, survival rate of MG63 cells was Icss than those under nomoxia conditions significantly. Exposed to hypoxia and GCV, DNA synthesis of MG63TK cells were inhibited, and cells accumulated in GoG, phase of cell cycles, and resulted in apoptosis and necrow. Conclusions: Hypoxia can enhance the selective killing effect and BSE of HSV-TIC/GCV system on human osteosarcoma cells and HRE-TIC/GCV system provides a new and effective means for gene therapy of osteosarcoma.