In 14p3HB transgenic mice, which carry three tandem copies of hepatitis B virus (HBV) DNA, the HBV DNA was significantly methylated and no viral proteins were produced. To analyze the causal relationship between hypermethylation and gene inactivity, 5-azacytidine was injected into the mice to demethylate HBV DNA. When postnatal 14p3HB mice were treated with the drug, hepatitis virus surface antigen was produced in these mice by 3 weeks of age, and the integrated HBV DNA of the liver was less heavily methylated. Our results suggest that injection of 5-azacytidine can be used to efficiently activate a silent transgene such as HBV DNA in transgenic mice.