The presence of antinuclear antibodies (ANA) has been described following hepatitis C virus (HCV). Very few studies have investigated the presence of anti-extractable nuclear antigens (ENA) in HCV infection. The aim of this study was to assess the prevalence of ENA antibodies in 100 patients with HCV infection compared to the prevalence of ENA antibodies in 100 healthy control patients. Sera from patients were tested for ENA using a multiplex microbead immunoassay. Sera positive for ENA were subsequently tested for ANA using an indirect immunofluorescence assay and titered if positive. Fourteen (14%) of the 100 patients with HCV infection had anti-ENA antibodies: four each showed anti-SSA antibodies and anti-dsDNA antibodies, three each had RNP antibodies and Scl-70 antibodies, and one each had anti-SSB, centromere B, Sm, and Sm/RNP antibodies. Ten of the 14 patients positive for anti-ENA were positive by indirect immunofluorescence staining (IFA) with titers ranging from 1:40 to 1:160. Five had antinuclear patterns, one had combined antinuclear and cytoplasmic patterns, and four only had a cytoplasmic pattern. Three of the 100 healthy control patients had ANA positive titers (1:80 and 1:320) and anti-ENA antibodies: one anti-Scl-70 and two anti-RNP. The prevalence of anti-ENA antibodies was significantly higher in the patients with HCV infections than in the healthy controls. Other studies of anti-ENA profiles in patients with HCV infection have identified similar patterns of positivity for anti-SSA, anti-SSB, anti-dsDNA, anti-RNP, anti- Sm/RNP, Scl-70, centromere B, and anti-Sm.