Some patients with chronic hepatitis B virus (HBV) infection are HB e antigen (HBeAg) negative, have circulating HBV particles, and often have especially severe chronic hepatitis. To test the hypothesis that the absence of HBeAg production may be due to a change in the nucleotide sequence of the pre-core region of the genome, 18 Greek and 3 non-Greek patients positive for HB surface antigen underwent direct sequencing of HBV-DNA amplified from sera. In 7 out of 8 HBeAg negative patients, two mutations (guanosine to adenosine) were found in the terminal two codons of the pre-core region, giving the sequence TAGGACATG. The remaining patient had the first mutation only. The sequence TGGGGCATG was found in 4 of 5 of the HBeAg positive patients. The first mutation results in a translational stop codon that is predicted to result in failure to produce HBeAg. The rest of the pre-core region in the HBeAg negative patients was otherwise homologous to that of the HBeAg positive patients and to known sequences.