We recently described the efficient assembly of hepatitis C virus (HCV) structural proteins into HCV-like particles (HCV-LPs) in insect cells. These noninfectious HCV-LPs have similar morphologic and biophysical properties as putative virions isolated from HCV-infected humans and can induce a broadly directed immune response in animal models. The HCV envelope proteins of HCV-LPs are presumably presented in a native, virion-like conformation and may therefore interact with antienvelope antibodies directed against conformational epitopes. In this study, HCV-LPs were used as capture antigens in an enzyme-linked immunosorbent assay (ELISA) to detect and quantify antibodies against HCV structural proteins in patients with acute and chronic hepatitis C. High titers of anti-HCV-LP antibodies were detected in patients chronically infected with HCV genotypes 1 to 6. In contrast to individuals with chronic hepatitis C, patients with acute self-limited hepatitis C displayed only a transient and weak seroreactivity against HCV-LPs. Patients with chronic HCV infection successfully treated with interferon demonstrated a gradual decline of anti-HCV-LP titers during or subsequent to viral clearance. Sustained interferon responders were characterized by significantly higher pretreatment levels of anti-HCV-LP antibodies as compared with nonresponders (P =.0001). In conclusion, HCV infection is associated with limited humoral immunity against the envelope proteins present on the HCV-LPs. An HCV-LP-based ELISA may be a useful diagnostic tool to distinguish acute hepatitis C from chronic HCV infection with exacerbation, and to predict viral clearance in response to interferon.