Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.