Two chimpanzees, 1535 and 1536, became persistently infected following inoculation with RNA transcripts from cDNA clones of hepatitis C virus (HCV). Analysis of the HCV genomes from both animals showed an accumulation of amino acid substitutions over time. The appearance of substitutions in the envelope genes was associated with increased antienvelope antibody titers. However, extensive mutations were not incorporated into hypervariable region 1 (HVR1). A comparison of the nonsynonymous substitution rate/synonymous substitution rate was made at various time points to analyze selective pressure. The highest level of selective pressure occurred during the acute phase and decreased as the infection continued. The nonsynonymous substitution rate was initially higher than the synonymous substitution rate but decreased over time from 3.3 × 10⁻³ (chimpanzee 1535) and 3.2 × 10⁻³ (chimpanzee 1536) substitutions/site/year at week 26 to 1.4 × 10⁻³ (chimpanzee 1535) and 1.7 × 10⁻³ (chimpanzee 1536) at week 216, while the synonymous substitution rate remained steady at ∼1 × 10⁻³ substitutions/site/year. Analysis of PCR products using single-stranded conformational polymorphism indicated a low level of heterogeneity in the viral genome. The results of these studies confirm that the persistence of infection is not solely due to changes in HVR1 or heterogeneity and that the majority of variants observed in natural infections could not arise simply through mutation during the time period most humans and chimpanzees are observed. These data also indicate that immune pressure and selection continue throughout the chronic phase.