HBV polymerase shares several regions of amino acid homology with other DNA-directed and RNA-directed polymerases. The amino acid residues Asp^(429), Gly^(518), Asp^(551), Lys^(585), and Gly^(641) in the conserved motifs A, B`, C, D, and E in the polymerase domain of HBV polymerase were mutated to alanine or histidine by in vitro site-directed mutagenesis. Those mutants were overexpressed, purified, and analyzed against DNA-dependent DNA polymerase activity and affinity for DNA binding. All those mutants did not show DNA-dependent DNA polymerase activities indicating that those five amino acid residues are all critical in DNA polymerase activity. South-Western analysis shows that amino acid residues Asp^(429) and Asp^(551) are essential to DNA binding, and Gly^(518) and Lys^(585) also affect DNA binding to a certain extent.