OBJECTIVE: To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted at telomerase-positive hepatocellular carcinoma. METHODS: Human liver cancer cell line HepGII and Hep3B, human embryonic kidney cell line 293, and normal human fibroblasts of the line BJ were cultured and added with adenoviruses CNHK300, ONYX-015 (55 000 protein deleted adenovirus), or wtAd5 (wild type 5) with different multiplicity of infection (MOI) for 7 days. 293 cells were used to measure the titer of the filial generation virus from different cells. The cell survival rate was calculated by MTT method 2, 4, 6, and 8 days after. Different cells were added with CNHK300 virus and then the E1A protein in the cytoplasm was measured by western blotting. Fluorescence microscopy was used to observe the CNHK300-EGFP proliferation after the cells were cultured and added with the virus for 1.5 hours. RESULTS: The replicative viruses CNHK300 and wtAd5 proliferated rapidly in HepGII and Hep3B cells since 24 hours after inoculation and proliferated 40625 and 65326 times respectively with a proliferation potential similar to that of the wild-type adenovirus and much higher than that of the ONYX-015 virus. CNHK300 of the MOI of 0.0002 killed half of the cancer cells, especially those of the line Hep3B, within 5 approximately 6 days, and CNHK300 virus of the MOI of 0.5 pfu/cell killed almost all the HepGII cells in the 8th day, with a killing power lower than that of the wild-type virus and higher than that of the ONYX-015 cells. The IC(50) was as low as MOI of 0.002 pfu/cell for the Hep3B cell and was as high as MOI of 100 pfu/cell for the BJ cell. CNHK300 was a less powerful killer of fibroblasts than wild-type virus. E1A expression was shown by western blotting in 293 cells and CNHK300-infected liver cancer cells, but not in the CNHK300-infected normal human fibroblasts. Fluorescence microscopy showed only isolated fluorescence-positive fibroblasts till the 10th day of infection, but obvious proliferation of CNHK300-EGFP virus since the 3rd day and fluorescence-positive cells in sheets by the 7th day, however, the fluorescent intensity was weakened since the 10th day. CONCLUSION: Tumor-selective adenovirus CNHK300 replicates in telomerase-positive liver cancer cells efficiently as well as wtAd5 and causes oncolysis, but has severely attenuated proliferation and cytolysis in normal cells.