The HBV RNA encapsidation signal epsilon (HBV epsilon) is an element essential for HBV virus replication. It is an RNA structure situated near the 5' end of the HBV pregenomic RNA. The structure consists of a lower stem, a bulge region, an upper stem and a tri-loop. The structure was determined and refined through enzymatic probing and NMR spectroscopy. The closure of the tri-loop was not predicted by RNA structure prediction programs but observed in the NMR structure. The regions shown to be critical for encapsidation of the RNA in the viral lifecycle are the bulge, upper stem and tri-loop which interact with the terminal protein domain of the HBV viral polymerase.