Replication-selective adenovirus has been reported to kill tumor cells and hold promise for cancer therapy. In this study, we constructed an E1B M(r) 55000-deleted adenovirus, designated Ad5WS1, and examined its cytolytic effect on human hepatocellular carcinoma (HCC) cell lines with various p53 status. The results show that Ad5WS1 lysed HCC cells lacking p53 transcription activity. However, this effect was not observed in cells harboring functional p53. Because loss of p53 transcription activity can be induced by binding to hepatitis B virus X protein (HBx), we generated HBx stable transfectants from Chang liver cells and examined their susceptibility to Ad5WS1-induced cytolysis. Expression of HBx in Chang liver cells changed the location of p53 from the nucleus to the cytoplasm, which mostly coincided with the location of HBx in the cytoplasm. Disruption of p53 transcription activity by HBx in Chang liver cells rendered them susceptible to infection with Ad5WS1. Furthermore, Ad5WS1 exerted antitumor effect, especially when combined with chemotherapeutic agent cisplatin, in BALB/c mice bearing HBx-expressing HCC. Our results suggest that E1B M(r) 55000-deleted adenovirus may have therapeutic potential for the treatment of HCC with loss of p53 transcription activity or with HBx expression.