To evaluate whether in vitro and in vivo transferring of Escherichia coli cytosine deaminase gene to a solid tumor will confer the sensitivity to the prodrug 5-fluorocytosine (5FC) on these cells, we constructed two replication-defective adenovirus vector in which the cytosine deaminase gene was driven by CAG promoter (Adex1CACD) and AFP gene 5'-flanking region (Adex1AFPCD), respectively. By transferring these two vectors to SMMC7721AFP(-) and HepG2 human hepatocellular carcinoma (HCC) cells in vitro, we found that Adex1CACD vector could effectively suppress SMMC7721AFP(-) and HepG2 cells growing in the presence of 5FC even if the infected cell is less to 20%, while Adex1AFPCD vector only conferred HepG2 cells sensitivity to 5FC. When Adex1CACD was directly injected into established subcutaneous SMMC7721AFP(-) tumors in nude mice receiving 5FC, the tumor growth was inhibited significantly, which was consistent with those in vitro results. Furthermore, the Adex1AFPCD plus 5FC suppressed SMMC7721AFP(+) tumor growth in vivo, but not SMMC7721AFP(-) tumor. The results suggested that the CAG promoter-controlled CD gene could effectively mediate the growth inhibition in different kinds of HCC combined with administration of 5FC, and the AFP promoter-controlled CD gene could only suppress the HCCs expressing high levels of AFP. Therefore, adenovirus-mediated tumor-specific gene transfer may be a potential strategy for local control of tumor growth.