We previously cloned mutant hepatitis B virus (HBV) genome, which had one nucleotide insertion in the X-C overlapping region resulting in a frameshift mutation of the X gene and fusion of the X and C genes. cDNA cloning from HepG2-K8, cell line producing mutant HBV particles, and sequence analysis, showed that the 0.9 kb polyadenylated RNA at nucleotide (nt) 1,950 can code for an elongated X protein of 193 amino acids (21 kDa) by the generation of an in-frame termination codon, TAA, at the poly(A) addition site. The elongated X open reading frame (ORF) was expressed in E. coli using the T7 inducible expression system. Western blotting with rabbit anti-X antisera showed a 21 kDa protein. The protein was purified from inclusion bodies through denaturation, DEAF-cellulose chromatography, heparin-agarose affinity chromatography, and renaturation procedures. Transcriptional activation activity of the elongated X protein was observed and indistinguishable from that of the wild-type X protein. These results suggest that the elongated X protein can function in place of the wild-type X protein in our mutant HBV that does not have a wild-type X-ORF.