The core promoter region of hepatitis B virus genomes regulates transcription of the precore and pregenomic mRNAs encoding hepatitis B e antigen (HBeAg) and core antigen that contain target epitopes for cytotoxic T lymphocytes. The prevalence and clinical significance of mutations in this region were investigated. DNA was extracted from six asymptomatic carriers positive for HBeAg, eight asymptomatic carriers positive for an anti-HBe antibody, and 24 patients with chronic liver disease. The core promoter and precore regions of hepatitis B virus genomes were amplified by polymerase chain reaction, and predominant sequences were determined by direct sequencing. Mutations were found in none of the HBeAg-positive asymptomatic carriers but in all of the anti-HBe-positive asymptomatic carriers and the patients with chronic liver disease. Especially, A to T mutations at nucleotide 1762 and G to A mutations at nucleotide 1764 were found in five anti-HBe-positive asymptomatic carriers, and 22 patients with chronic liver disease. These two mutation hot spots were located within binding sites of the nuclear factors, and nucleotide 1762 was also involved in the A, T rich sequence that is located 28 base pairs upstream of the precore mRNA initiation site. Serum HBeAg and DNA polymerase levels were significantly lower in patients with these mutations than those without these mutations, and five individuals with these mutations were positive for anti-HBe despite the absence of the precore stop codon mutation. These mutants may be selected by host immune response to HBeAg and/or core antigen.