To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.