Aim: Mutant HBV strains have been reported to have relation to the various clinical manifestations in chronic HBV infection. However, mutations in the polymerase (pol) gene, which is important in viral life cycle, has not been fully elucidated. We performed a sequencing analysis in the terminal protein (TP) region, the amino-terminal domain of the pol gene, on patients with chronic HBV infection. Methods: The subjects were six patients with chronic HBV infection (duration of follow-up, 3.5-10.0 yr). At the beginning of follow-up, serum ALT was high (92-1406 IU/l), and HBeAg was positive in all the six patients. Serum HBV DNA titers determined by PCR with end-point dilution ranged from 10 5 to 10 7 . During follow-up, all of them showed the sustained ALT normalization and HBeAg clearance. At the end of follow-up, serum HBV DNA became undetectable in one patients, and HBV DNA titers ranged from 1 to 10 in the remaining five patients. Sera obtained at both the beginning and the end (or 1 yr before end) of follow-up were used for the analysis. TP region was amplified from serum samples by nested PCR. The PCR products were subcloned and sequenced by the dideoxy chain termination method. Results: The net nucleotide substitutions rate in the TP region on each of the six patients ranged from 3.90 10 - 4 to 9.63 10 - 3 (mean 2.53 10 - 3 ) site - 1 year - 1 , and was 10- to 100-fold higher than those previously reported by a few researchers. Two to five amino acid substitutions were observed in five of the six patients. Two novel amino acid substitutions (W to C at codon 52 in one patient and T to A at codon 60 in another patient) changed both predicted hydropathic character and secondary structure of the pol protein. Conclusion: Our findings indicate that substantially high rate of mutations may occur in the TP region during the course of chronic HBV infection, and that some of amino acid substitutions might cause the dysfunction of the pol protein, resulting in the decline of viral replicative levels.