OBJECTIVE: To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). METHODS: A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by cloning and sequencing analysis. RESULTS: The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 as well were constructed successfully as confirmed by sequence analysis with RFLP. CONCLUSION: The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.