Hepadnavirus replication occurs in hepatocytes in vivo and in hepatoma cell lines in cell culture. Hepatitis B virus (HBV) replication can occur in nonhepatoma cells when pregenomic RNA synthesis from viral DNA is activated by the expression of the nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X receptor a (RXRa) plus peroxisome proliferator-activated receptor a (PPARa) heterodimer. Nuclear hormone receptor-dependent HBV replication is inhibited by hepatocyte nuclear factor 3 (HNF3). In contrast, HNF3 and HNF4 support duck hepatitis B virus (DHBV) replication in nonhepatoma cells, whereas the RXRa-PPARa heterodimer inhibits HNF4-dependent DHBV replication. HNF3 and HNF4 synergistically activate DHBV pregenomic RNA synthesis and viral replication. The conditions that support HBV or DHBV replication in nonhepatoma cells are not able to support woodchuck hepatitis virus replication. These observations indicate that avian and mammalian hepadnaviruses have distinct transcription factor requirements for viral replication.