Background: The hepatitis C virus (HCV) mutates within human leucocyte antigen (HLA) class I restricted immunodominant epitopes of the non-structural (NS) 3/4A protease to escape cytotoxic T lymphocyte (CTL) recognition and promote viral persistence. However, variability is not unlimited, and sometimes almost absent, and factors that restrict viral variability have not been defined experimentally. Aims: We wished to explore whether the variability of the immunodominant CTL epitope at residues 1073–1081 of the NS3 protease was limited by viral fitness. Patients: Venous blood was obtained from six patients (four HLA-A2+) with chronic HCV infection and from one HLA-A2+ patient with acute HCV infection. Methods: NS3/4A genes were amplified from serum, cloned in a eukaryotic expression plasmid, sequenced, and expressed. CTL recognition of naturally occurring and artificially introduced escape mutations in HLA-A2-restricted NS3 epitopes were determined using CTLs from human blood and genetically immunised HLA-A2-transgenic mice. HCV replicons were used to test the effect of escape mutations on HCV protease activity and RNA replication. Results: Sequence analysis of NS3/4A confirmed low genetic variability. The major viral species had functional proteases with 1073–1081 epitopes that were generally recognised by cross reactive human and murine HLA-A2 restricted CTLs. Introduction of mutations at five positions of the 1073–1081 epitope prevented CTL recognition but three of these reduced protease activity and RNA replication. Conclusions: Viral fitness can indeed limit the variability of HCV within immunological epitopes. This helps to explain why certain immunological escape variants never appear as a major viral species in infected humans.