The existence of hepatitis C virus (HCV) proteins encoded by alternate reading frames overlapping the core-encoding region has been suggested. Several mechanisms of production have been postulated, and the functions of these proteins in the HCV life cycle remain unknown. We analyzed cases of seroconversion to an alternate reading frame protein in a group of 17 patients infected by one of the two HCV genotype 1b strains during an outbreak in a hemodialysis unit. Three patients seroconverted, and antibodies were transiently detected in another patient. Three of these patients were infected by one of the two HCV strains, whereas the strain infecting the remaining patient could not be identified. Quasispecies sequence analysis of the core-coding region showed no differences in the core or ؉1 reading frame sequences that could explain alternate reading frame protein seroconversion in some but not all of the patients infected by one of the HCV strains, and no such difference was found between the two strains. Because differences in the structure of RNA elements could play a role in frameshift events, we conducted a predictive analysis of RNA folding. No difference was found between the patients who did and did not seroconvert to alternate reading frame protein. Conclusion: Our findings prove that alternate reading frame proteins can be produced during acute HCV infection. However, seroconversion does not occur in all patients for unknown reasons. Alternate reading frame protein could be generated by minority quasispecies variants or variants that occur transiently. (HEPATOLOGY 2009;49: 1449-1459.) H epatitis C virus (HCV), a member of the Flaviviridae family, has a single-stranded positive RNA genome of approximately 9,600 nucleotides. The genome is composed of a 5Ј noncoding region, a long open reading frame (ORF) encoding the precursor polyprotein of about 3,000 amino acids, and a 3Ј noncoding region. The ORF encodes 10 proteins that are generated through processing of the precursor polyprotein by cellular and viral proteases. HCV proteins comprise structural proteins (core protein and the two envelope glycoproteins E1 and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that have various functions in the HCV lifecycle. 1,2 In 2001, Walewski et al. 3 used computerized sequence analysis to map dual-use regions in HCV coding sequences. The results strongly suggested the existence of a new HCV antigen encoded, at least in part, by an alternate, ϩ1 reading frame overlapping the core encoding region. This was further supported by the observation of a single stop codon in the ϩ1 reading frame of reference HCV strains at positions that depended on the HCV genotype. 4 The detection of specific antibodies targeting this protein in patients with chronic hepatitis C via both enzyme immunoassay and western blotting strongly suggests that alternate reading frame proteins (ARFPs) are produced during HCV infection. 3,5 Abbreviations: ALT, alanine aminotransferase; ARFP, alternate reading frame protein; ELISA, enzyme-linked immunosorbent assay; HCV, hepatitis C virus; ORF, open reading frame; PCR, polymerase chain reaction.