Cytotoxic T lymphocytes (CTLs) may play a role in host defence against hepatitis C virus (HCV) infection, and HCV-specific CTL epitopes may be included in vaccines to induce protective CTLs. We identified three new epitopes within the HCV nucleoprotein recognized by CTLs. HCV nucleoprotein residues 28–37 are the minimal epitope recognized by CTLs in association with the class I human leukocyte antigen B60, and epitopes in HCV nucleoprotein residues 111–130 and 161–180 are both recognized by CTLs in association with the class II human leukocyte antigen DRB1*08032.