An attempt was made to infect primary duck hepatocyte cultures with duck hepatitis B virus in vitro in order to clarify the biology of hepatitis B virus. Livers of ducklings, 0 to 17 days posthatch, without viremia were digested ex situ by perfusion of collagenase solution through the portal or hepatic vein. Homogeneous hepatocyte suspensions were seeded into plastic dishes in L-15 medium containing 10−8M insulin, 2 × 10−8M glucagon and 10−8M dexamethasone and were subsequently inoculated with sufficient numbers of duck hepatitis B virus. As a result, duck hepatitis B virus multiplication started weakly on Day 2, gradually increased and reached the maximum level approximately on Day 10 postinoculation. Viral replication was revealed by duck hepatitis B virus DNA in the cell pellet and in the culture medium and duck hepatitis B virus DNA-specific transcripts in the cell pellet. Immunostaining demonstrated duck hepatitis B virus core antigen in approximately 10% of cultured hepatocytes, and an increase in numbers of positive cells was not observed with time for up to 18 days of culture. Viral particles were found within the endoplasmic reticulum, and the inoculation of culture medium provoked viremia in the ducklings. The age of ducklings did not influence the numbers of cells infected. The in vitro infection system was similar to the in vivo one; however, the former seemed to be age-independent and to allow replication of duck hepatitis B virus in the limited number of hepatocytes.