The work is to construct a hypoxia-activated adenovirus vector expressing suicide gene BCD and to evaluate anti-tumor effects of the hypoxia-targeted suicide gene therapy system with or without combination radiotherapy. The recombinant adenovirus Ad-5HRE/hCMVmp-BCD was generated by the COS-TPC methods. Hela cells were treated with Ad-5HRE/hCMVmp-BCD infection, followed by incubated with 5FC-containing medium under the aerobic conditions or hypoxic conditions for 24h. Cell growth inhibition was determined by MTS assay. Tumor xenografts were established by injecting Hela cells s.c into the right leg of nu/nu BALB/c mice. The tumor-bearing mice were given Ad-5HRE/hCMVmp-BCD- mediated gene therapy or combination therapy with radiotherapy. Western blot analysis clearly indicated that significant hypoxia-induced expression of BCD protein after Hela cells were infected with Ad-5HRE/hCMVmp-BCD. The actual amounts and the ratio of the BCD expression under aerobic and hypoxic conditions were increased with increasing MOI. MTS assay results indicated that hypoxia-induced sensitization to 5FC. Under the hypoxic incubation the IC,50, is 20 folds lower than that of the aerobic condition. The increased cytotoxicity at each MOI of Ad-5HRE/hCMVmp-BCD infection with increasing 5FC concentration was also observed. Tumor growth delay assay results showed that with single radiation or fractionated radiation, the Ad-5HRE/hCMVmp-BCD+5FC +radiation group produced the longest times to tumor regrowth and average tumor doubling compared with the various control treatment groups. In vitro and in vivo studies demonstrated the combination of Ad-5HRE/hCMVmp-BCD and prodrug 5-FC can obviously inhibit the growth of Hela cells. Using the mouse xenografts model, we demonstrated the Ad-5HRE/hCMVmp-BCD and 5-FC can enhance the antitumor effects of conventional radiation therapy.