Hepatitis C virus (HCV)-specific CD4+ and CD8+ T cell responses were investigated using a panel of 728 overlapping peptides spanning the whole HCV genome in 47 HCV mono-infected and 26 HIV/HCV co-infected individuals using the IFN-gamma ELISPOT assay and flow cytometry. The frequency of HCV-specific T cell responses was similar (approximately 40%) in both groups, but the breadth of the T cell responses tended to be reduced in HIV/HCV co-infected individuals. Of interest, 23 new HCV-derived epitopes were identified, and CD4+ HCV-specific T cell responses were detected overall in a proportion similar to CD8+ T cell responses. A tendency towards a dominant CD8+ T cell response was associated with HIV/HCV co-infection. HCV-specific CD8+ T cells secreted both IL-2 and IFN-gamma, although a reduction in the percentage of IL-2/IFN-gamma-secreting cells was observed in HIV/HCV co-infected individuals. The increase in CD4+ T cell counts after antiretroviral therapy in HIV/HCV co-infected individuals was not associated with restoration of HCV-specific T cell responses. Altogether, these results provide new insights into the characterization of HCV-specific T cell responses in HCV mono-infected and HIV/HCV co-infected individuals.