The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.Hepatitis C virus (HCV) is an important cause of morbidity and mortality worldwide (1-3). Infection with HCV becomes chronic in Ͼ80% of the cases and is a major cause of liver cirrhosis (4) and hepatocellular carcinoma (5). Although the development of a broadly reactive vaccine would be the most effective method for its control, concerns have been raised because of the high degree of genetic heterogeneity of HCV (6) and the lack of protective immunity against reinfection (7,8) or superinfection (9, 10) documented both in humans and in chimpanzees. Viral isolate-restricted neutralizing antibodies against HCV have been demonstrated recently in infected individuals (11, 12), but their molecular target is presently unknown.Several observations have suggested that the hypervariable region 1 (HVR1) could be involved in the neutralization of HCV. This assumption is based on the fact that the HVR1, which is located at the N terminus of the envelope glycoprotein 2 (E2) gene and consists of 34 amino acids spanning map position 384-414 (13), is the most variable region of the HCV genome (14, 15), contains linear epitopes that are recognized by patients' antibodies (16-22) and mutates rapidly in vivo (23)(24)(25)(26), suggesting that it is under the selective pressure of the host immune system. This hypothesis is further substantiated by the lack of variability in the HVR1 observed in an agammaglobulinemic patient over a period of 2.5 years (27). Recent data obtained in vitro suggest that antibodies, present in human sera and directed against the HVR1 as well as against the E2 protein of HCV, can prevent the binding of HCV to cells (28,29). The potential importance of the HVR1 for HCV neutralization is also underscored by the analogy with the V3 loop of human immunodeficiency virus, w...