The HBx-derived, HLA-A2.1 restricted peptides, XEP-3, XEP-4, and XEP-6, induced activation of specific CTLs from patients with HBV in vitro. XEP-6 peptide induced the strongest response among the three peptides in CTLs from the blood samples of patients that were HBsAg positive. It was not clear whether the stage of disease (chronic infection, cirrhosis or hepatoma) was related to the responsiveness of the CTLs to each peptide. We vaccinated HLA-A2/K(b) transgenic mice with these peptides encapsulated in pH-sensitive liposomes at various concentrations and tested their ability to protect against challenge with rVV-HBx. Mice immunized with encapsulated peptides were protected against viral challenge whereas those immunized with empty liposomes were not. In general, 5 micro g of each peptide per head inoculation was sufficient to give protection after 2 weeks. After 3 weeks, this protective effect was increased. This effect of time was more important on the level of protection than the initial dose of the peptide. To explain the protective effect, IFN-gamma secreting CD8(+) cells isolated from mice 3 weeks after immunization were analyzed ex vivo. There was little dose dependency of peptide on IFN-gamma secretion except for XEP-3. The variations in the results may reflect the chemical properties of the peptides, such as solubility and binding affinity. In conclusion, epitope peptides derived from HBx can induce specific CTL activation and lead to cellular immunity in vitro and in vivo by inducing the peptide-specific CD8(+) CTLs. Thus, pH-sensitive liposomes increase the immune response following immunization with a peptide vaccine. This could be used for the treatment of HBV-related disease.