In an attempt to determine the effect of aflatoxin B1 (AFB) intoxication on livers with duck hepatitis B virus (DHBV) infection, domestic ducks were given 0.1 mg of AFB/kg body weight twice a week for a maximum period of 54 weeks employing various experimental designs. The ducks were infected with DHBV by i.v. inoculation of DHBV-positive sera within 24 h posthatch. The livers were examined histologically, immunohistochemically, and ultrastructurally, and the livers and sera were examined by molecular hybridization for DHBV DNA. AFB administration induced hepatocellular necrosis and marked biliary cell proliferation of the periportal areas, and finally liver cirrhosis. On short-term administration, the hepatocytes of DHBV-infected livers revealed a marked increase in incomplete particles of DHBV by immunostaining and electron microscopy, as compared to those without its administration. Long-term AFB administration provoked frequent nodular or cirrhotic changes. There was no significant increase in frequency of these changes in DHBV-positive ducks as compared to DHBV-negative ones. AFB administration induced hepatocellular carcinoma (HCC) in one DHBV-positive duck and in two DHBV-negative ducks. The HCC and cirrhotic livers revealed extrachromosomal but no integrated form of DHBV DNA by Southern blot hybridization analysis. Immunostaining demonstrated a heterogeneous distribution of DHBV from area to area in nodular and cirrhotic livers. Thus, AFB intoxication provoked various liver disorders independent of DHBV infection, and neither a cocarcinogenic effect of AFB and DHBV nor integration of viral DNA into the genome of neoplastic and nonneoplastic tissues was observed in the present experiments. Generally speaking, DHBV infection did not appear to accelerate hepatic disorders induced by AFB intoxication. However, AFB administration altered the DHBV in the liver in terms of its amount and distribution.