Hepadnaviral preS proteins are important in viral entry but their exact roles are incompletely defined. A major function of viral envelope proteins is host range determination.To assign functions to preS and S in entry, we first constructed replication-competent, overlength (1.5-mer) plasmids from both HHBV (heron hepatitis B virus) and DHBV (duck hepatitis B virus). Into each, we introduced (i) a stop codon in preS (preS-S+ vector), or (ii) an ATG to ACG change in the S initiator (preS+S- vector): neither mutation disrupts the polymerase ORF. Cotransfection of LMH cells with pairwise combinations of these plasmid gave rise to virions bearing different combinations of preS and S proteins from HHBV or DHBV. Progeny virions were then tested for their ability to infect primary duck hepatocyte (PDH) cells. Only when preS polypeptides were derived from DHBV did progeny grow in PDH; envelopes bearing HHBV preS plus DHBV S proteins could not mediate efficient entry into PDH.To further define host-range determinants, we constructed a series of expression vectors encoding chimeric DHBV/HHBV envelope proteins. These vectors lack C and P expression and can only express the chimeric preS and S proteins. Each chimera was then used to pseudotype an HHBV 1.5-mer genome bearing an S stop codon, by cotransfection into LMH cells. The pseudotype bearing DHBV preS precisely fused to HHBV S showed efficient infectivity for PDH cells.