Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.