One hundred and seventeen ducklings, 42 inoculated with duck hepatitis B virus (DHBV) 2 days after hatching and 55 connatally infected, were studied over a 6-month period in parallel with 20 ducklings without DHBV infection. Using immunohistochemical, in situ and blot hybridization analyses, the natural course of hepatic and extrahepatic infection was examined. DHBV infection started in the liver 2-4 days post-inoculation. There, DHBV was found not only in hepatocytes, but also in bile duct epithelial cells. Further, DHBV infection occurred in exocrine and endocrine pancreas (beginning 6-10 days and 20 days post-inoculation, respectively) and in germinal centers of the spleen (beginning 8 weeks post-inoculation). Occasionally viral DNA was also found in kidney glomeruli. Using strand-specific RNA probes, viral DNA in pancreas and spleen was clearly demonstrated to be replicating intermediates. Hepatic and extrahepatic infection with DHBV was not associated with histologic inflammation or pathologic changes in these tissues or the liver. These data indicate that DHBV can infect cells other than hepatocytes. The biological significance of non-hepatocyte infection for the life-cycle of the virus and its potential significance for viral persistence remain to be determined.