During chronic hepatitis B virus (HBV) infection, virus persistence relies on the maintenance of a pool of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. To achieve this, HBV DNA has to be transported from the cytoplasm to the nucleus. By carrying out subcellular fractionation experiment, both of the relaxed‐circular (RC) and single‐stranded (SS) HBV DNA were found in the cytoplasm whereas only RC form could be detected in the nucleus of a hepatoblastoma cell line (HepG2) stably producing HBV. This fraction of nuclear RC viral DNA was clearly demonstrated in the G₁ but not S phase of synchronized HepG2 cells. Conversely, the relative amount of cytoplasmic RC viral DNA in the S phase was larger than that in the G1 phase. Although no cccDNA could be detected in HepG2 cells without synchronization, an increasing amount of cccDNA in the nucleus was demonstrated after prolonged incubation of the cells in aphidicolin. Finally, by undertaking in situ hybridization using a probe specific to plus‐strand HBV DNA, nuclear viral DNA was detected predominantly in the G1 phase of HepG2 cells. In summary, the results indicated that only RC but not SS form of HBV DNA was localized to the nuclei of HepG2 cells. The nuclear localization occurred preferentially in the G₁ but not S phase and prolonged treatment with aphidicolin resulted in accumulation of nuclear cccDNA. J. Med. Virol. 55:42–50, 1998. © 1998 Wiley‐Liss, Inc.