Mutations in the hepatitis B virus (HBV) core gene may influence disease activity by altering immune recognition sites or level of virus replication. Sera from 69 Chinese patients with chronic HBV infection were analyzed by direct sequencing of polymerase chain reaction amplification of HBV DNA to determine the frequency and location of naturally occurring HBV core gene mutations. All but one patient had nucleotide changes, and 44 (64%) patients had at least one amino acid change (mean, 3.7; range, 1-13) when compared with published sequences. Multiple regression analysis showed that the frequency of core gene mutations was significantly associated with precore stop-codon mutation, hepatitis B e antigen negativity, and active liver disease, but not patients' age. The mean number of amino acid changes/patient for hepatitis B e antigen (HBeAg)-positive patients with elevated versus normal aminotransferase levels were, respectively, 2.8 ± 0.4 and 0.6 ± 0.2. The corresponding values for HBeAg-negative patients were, respectively, 5.0 ± 1.2 and 6.0 ± 1.5. Thirteen patients were serially studied, the mean rates of amino acid substitution in HBeAg-positive patients who did or did not clear HBeAg during follow-up were 5.7 ± 0.8 and 0 per codon/yr. Most of the mutations were clustered in the middle of the core gene that harbor several major B- and helper T-cell epitopes. Very few mutations were found in the C-terminal part of the core gene. In summary, mutations in the core gene can be frequently detected in patients with chronic HBV infection. These mutations occur predominantly around the time of HBeAg clearance when liver disease is most active. (HEPATOLOGY 1995; 22:50–60).