Interferons control viral replication and the growth of some malignant tumors. Since systemic application may cause severe adverse effects, tissue-specific expression is an attractive alternative. Liver-directed interferon gene therapy offers promising applications such as chronic viral hepatitis B or C or hepatocellular carcinoma and thus needs testing in vivo in suitable animal models. We therefore used the Tet-On system to regulate gene expression in adenoviral vectors, and studied the effect of liver-specific and regulated interferon gamma expression in a mouse model of chronic hepatitis B virus (HBV) infection. In a first generation adenoviral vector, genes encoding for firefly luciferase and interferons alpha, beta or gamma, respectively, were coexpressed under control of the bidirectional tetracycline-regulated promoter P(tet)bi. Liver-specific promoters driving expression of the reverse tetracycline controlled transactivator ensured local expression in the livers of HBV transgenic mice. Following gene transfer, we demonstrated low background, tight regulation and a 1000-fold induction of gene expression by doxycycline. Both genes within the bidirectional transcription unit were expressed simultaneously, and in a liver-specific fashion in cell culture and in living mice. Doxycycline-dependent interferon gamma expression effectively controlled HBV replication in mice, but did not eliminate HBV transcripts. This system will help to study the effects of local cytokine expression in mouse disease models in detail.