Hepatitis C virus (HCV) encodes ten classic proteins as well as a newly discovered alternative reading frame protein (ARFP) whose synthesis originates from the core region by a +1 frameshift. ARFP is produced by all HCV genotypes, but its function remains unknown. Although the immunogenicity of genotype 1- and 2-derived ARFP in infected hosts has been reported, no information is available for genotype 3-encoded ARFP. HCV genotype 3 core/ARFP region was PCR amplified, cloned, and sequenced. Recombinant ARFP and peptides were employed in ELISAs with patient serum samples. The effect of peptides on peripheral blood mononucleocytes (PBMCs) was also studied. DNA cloning and sequencing of HCV genotype 3 strain (PKHCV3) revealed it to encode 160 aa ARFP, which harbors a C-terminal extension of 36 aa. Serum from 74 of 88 patients (84%) contained rARFP-reactive antibodies. Peptide ELISAs showed that all regions of rARFP were immunogenic, with peptide F7 (DSLSPRRAGAKAGPGLSPGT) being the most immunodominant. When incubated with PBMCs from HCV-infected individuals, F7 stimulated the production of TNFα and IL10. PKHCV3-derived ARFP encodes a 160 aa protein and antibodies against its entire length are found in 84% of all genotype 3-infected subjects. Peptide ELISAs revealed F7 to be highly immunogenic and capable of eliciting impressive T-cell responses.