Previously, we reported that a 7-mer HLA-A11-restricted peptide (YVNTNMG) of hepatitis B virus (HBV) core Ag (HBcAg(88-94)) was associated with heat shock protein (HSP) gp96 in liver tissues of patients with HBV-induced hepatocellular carcinoma (HCC). This peptide is highly homologous to a human HLA-A11-restricted 9-mer peptide (YVNVNMGLK) and to a mouse H-2-K(d)-restricted 9-mer peptide (SYVNTNMGL). To further characterize its immunogenicity, BALB/c mice were vaccinated with the HBV 7-mer peptide. It was found that a specific CTL response was induced by the 7-mer peptide, although the response was approximately 50% of that induced by the mouse H-2-K(d)-restricted 9-mer peptide, as detected by ELISPOT, tetramer, and (51)Cr release assays. To evaluate the adjuvant effect of HSP gp96, mice were coimmunized with gp96 and the 9-mer peptide, and a significant adjuvant effect was observed with gp96. To further determine whether the immune effect of gp96 was dependent on peptide binding, the N- and C-terminal fragments of gp96, which are believed to contain the putative peptide-binding domain, were cloned and expressed in Escherichia coli. CTL assays indicated that only the N-terminal fragment, but not the C-terminal fragment, was able to produce the adjuvant effect. These results clearly demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment CTL response against HBV infection and HCC.