Accurate translocation of the polymerase-linked oligomer to the acceptor site (DR1*) in reverse transcription is crucial for maintaining the correct size of the hepatitis B virus (HBV) genome. Various sizes of foreign sequences were inserted at different sites of the HBV genome, and their effects on accurate translocation of polymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in these insertion mutants: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicated that the minus strand of RC and type I DL form was elongated from DR1*, while the minus strand of the type II DL form was elongated from multiple internal acceptor sites (IAS), such as IAS2. These IASs were also found to be used by wild-type HBV but with a very low frequency. Mutation of IAS2 by base substitution abrogated polymerase-linked oligomer transferring to IAS2, demonstrating that base pairing also plays an important role in the function of IAS2 as a polymerase-linked oligomer acceptor site. Data obtained from our insertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site and the acceptor is important. The polymerase-linked oligomer prefers to translocate to an acceptor, DR1* or IAS2, which are ca. 3.2 kb apart. However, it will translocate to both DR1* and IAS2 if they are not located 3.2 kb apart. These results suggest that the polymerase-linked oligomer may be able to scan bidirectionally for appropriate acceptor sites at a distance of 3.2 kb. A model is proposed to discuss the possible mechanism of polymerase-linked oligomer translocation.