Hepatitis B virus (HBV) possesses a 3.2-kb partially double-stranded DNA genome that is generated inside the nucleocapsid by the reverse transcription of the 3.5-kb pregenomic viral transcript. The initial steps in viral replication involve the recognition of an encapsidation signal termed epsilon (epsilon) at the 5'-end of the pregenomic RNA by the HBV polymerase. The polymerase-bound pregenomic RNA is subsequently incorporated into an immature nucleocapsid particle and minus-strand HBV DNA synthesis is initiated utilizing the bulge region of epsilon as a template and a tyrosine residue in the amino-terminal region of the polymerase as a primer. Three nucleotides complementary to the 3'-end of the bulge region of epsilon are synthesized and subsequently translocated with the polymerase molecule to the acceptor site located in the DR1 sequence present at the 3'-end of the pregenomic RNA. Using mutagenesis analysis, a sequence element designated phi (phi) located upstream of the 3' DR1 sequence has been identified that is complementary to epsilon and is important for efficient viral replication. This element may bring the 3' DR1 sequence into proximity with the three nucleotide primer synthesized at the bulge of epsilon and facilitate primer translocation to the 3' DR1 acceptor sequence. Sequence elements with similar proximity to the 3' DR1 sequences and complementarity to epsilon are present in the woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV), suggesting the phi regulatory element may be phylogenetically conserved due to its functional importance in hepadnavirus minus-strand DNA synthesis.