The expression of hepatitis B virus (HBV) DNA in the liver was studied by nonisotopic in situ hybridization and correlated with liver histology, different phases in the natural evolution of chronic hepatitis B, and hepatic expression of HBV antigens in 251 Chinese patients with chronic HBV infection. A good correlation was found between the detection of HBV-DNA by in situ hybridization and serum HBV-DNA (P < .01). Chronic active hepatitis had the highest HBV-DNA detected in cytoplasm and nuclei, compared with livers showing minimal change, chronic persistent hepatitis, cirrhosis, and hepatocellular carcinoma. HBV-DNA in cytoplasm exceeded HBV-DNA in nucleus in all patients except in livers with hepatocellular carcinoma. Hepatic HBV-DNA correlated with disease activity (P < .02) and the correlation was highly significant with intralobular activity (P < .001). Patients in the early viral replicative phase of infection had higher levels of cytoplasmic and nuclear HBV-DNA compared with the late viral nonreplicative phase. Cytoplasmic and nuclear HBV-DNA correlated with hepatic expression of HBcAg and HBsAg (P < .05 in both cases), but not with HBeAg. These data indicate that hepatic expression of HBV-DNA follows the natural history of chronic HBV infection and is associated with active liver disease.