Liver histology in chronic hepatitis B is marked by inflammatory infiltration involving the peripheral zones. The cause of such cellular infiltration remains unknown. The aim of the present study was to investigate the amounts of intrahepatic hepatitis B virus (HBV) DNA separately in the peripheral and central zones, using laser capture microdissection coupled with real-time quantitative polymerase chain reaction. Fourteen patients with chronic hepatitis B were included in the study. Liver biopsy samples were taken and hepatocytes were microdissected separately from peripheral and central zones. DNA was extracted from hepatocytes in each zone and evaluated the amounts of HBV-DNA. Immunohistochemical study for hepatitis B core antigen (HBcAg) was also performed. The amounts of total intrahepatic HBV-DNA in patients positive for hepatitis Be antigen (HBeAg) were greater than those in HBeAg-negative patients. There was no difference in HBV-DNA between the peripheral and central zones. Immunohistochemistry also showed that HBcAg-positive cells were distributed homogeneously in the hepatic lobules. In patients with peripherally predominant HBV-DNA, the serum alanine aminotransferase (ALT) level was lower than in patients with centrally predominant HBV-DNA. HBV-DNA was distributed homogeneously in the hepatic lobules. In patients with lower amounts of HBV-DNA in the peripheral zone, the serum ALT level tended to be higher than in other patients.