The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-epsilon interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-epsilon interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific epsilon binding. We now report that Hsp90 is also required for HBV RT-epsilon interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-epsilon interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-epsilon binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-epsilon interaction in HBV, as in DHBV. Specific epsilon binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV epsilon, but not the DHBV epsilon, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of epsilon was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-epsilon interaction and chaperone activation.