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.