Summary. We have previously increased the efficiency of hepatitis B virus (HBV) infection of human hepatocytes in vitro by using polyethylene glycol. After further documenting by neutralization experiments, this in vitro infection, we used this model to define new culture conditions that would maintain stable episomal replication for several weeks. We found that in the presence of 10% porcine serum and 2% dimethyl sulphoxide (DMSO), high-density cultures survived more than 3 months, while addition of hydrocortisone instead of DMSO resulted in survival of less than 1 month. HBV episomal replication was maintained without any evidence of viral integration into the host genome. The maintenance of HBV replication was demonstrated by: first, stability of the covalently-closed-circular DNA in the nucleus and relaxed circular and single-stranded replicative intermediates in the cytoplasm; second, detection of two major transcripts of 3.5 and 2.1–2.4 kb corresponding to the pregenomic and surface genes respectively; and third, continuous secretion of mature viral particles in the supernatant of infected cells. We showed that under these culture conditions, hepatocytes were blocked in the G1 phase of the cell cycle and did not spontaneously proliferate. Upon hepatocyte growth factor (HGF) stimulation, however, the ability of hepatocytes to divide was demonstrated and was compared in infected and non-infected cells. No change in proliferative capacity and no variation in c-myc and c-jun levels could be found. Hepatocyte survival was not modified in infected cells, confirming that HBV is not cytopathic for normal human hepatocytes. These new culture conditions represent substantial progress in the study of HBV-host cell interactions.