CD8(+) T-cell responses are central for the resolution of hepatitis C virus (HCV) infection, and viral escape from these CD8(+) T-cell responses has been suggested to play a major role in HCV persistence. However, the factors determining the emergence of CD8 escape mutations are not well understood. Here, the first identification of four HLA-A26-restricted CD8(+) T-cell epitopes is reported. Of note, two of these four epitopes are located in the NS3/4A and NS5A/5B cleavage sites. The latter epitope is targeted in all (three of three) patients with acute, resolving HCV infection and in a relatively high proportion (four of 14) of patients with chronic HCV infection. Importantly, the epitope corresponding to the NS5A/5B cleavage site is characterized by the complete absence of sequence variations, despite the presence of functional virus-specific CD8(+) T cells in our cohort. These results support previous findings that showed defined functional constraints within this region. They also suggest that the absence of viral escape may be determined by viral fitness cost and highlight an attractive target for immunotherapies.