Tissues from a 10-week-old Pekin duck, experimentally infected at 1 day of age with duck hepatitis B virus (DHBV), were examined for the presence of replicative levels of DHBV DNA by in situ and Southern blot hybridization. Hepatocytes, pancreatic lymphoid follicle, exocrine, and endocrine cells, and splenic mononuclear cells all contained DHBV DNA localized predominantly to the cytoplasm of infected cells. Duck hepatitis B surface antigen distribution in the same tissues correlated well with the presence of DHBV DNA in many of these cells. In hepatocytes and pancreatic islet cells, 60% of DHBV DNA was present as single-stranded DNA, indicating the likelihood of ongoing virus replication in these cell types and providing further evidence that hepadnavirus DNA replication occurs largely within the cell cytoplasm. In contrast, DHBV in mononuclear cells within splenic germinal centers was wholly double-stranded, suggesting that limited, if any, DHBV DNA replication was occurring in this cell type. These data provide further information about the pathogenesis and cell-specific sites of DHBV infection.