Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-α2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2′-5′ oligoadenylate synthetase/ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.