Previous efforts for biochemical study of the human hepatitis B virus (HBV) DNA polymerase have been limited by its tight association with viral nucleocapsids. We report here that the soluble DNA polymerase from HBV particles was obtained by low pH treatment of the viral particles followed by incubation with small amounts of subtilisin. By these treatments, the approximately 100-kDa band in the activity gel assay was gradually converted to approximately 70 kDa, which subsequently showed reverse transcriptase activity on several exogenous templates. The single approximately 70-kDa active band, which did not show any DNA polymerase activity in endogenous reaction, was eluted through DEAE-Sepharose chromatography. These results suggest that the approximately 100-kDa protein, most likely the product of HBV Pol open reading frame, is tightly associated with viral nucleocapsids, and the approximately 70-kDa protein, the proteolytic cleavage product of approximately 100-kDa enzyme, is solubilized from viral particles as an active enzyme on exogenous templates.