An association has been reported between mutations in the amino acid residues 2209-2248 of the nonstructural protein 5A (NS5A) gene (interferon-sensitivity determining region [ISDR]) and interferon efficacy in hepatitis C virus (HCV)-1b infection. This relationship was analyzed in chronic HCV-2 infection. Forty patients with HCV-2a and 35 with HCV-2b were treated with interferon alfa for 6 months with a total dose of 468 to 860 million units. Pretreatment NS5A sequences were determined by direct sequencing. A higher complete and sustained response rate was observed in HCV-2a than in HCV-2b (70% vs. 34%; P = .003). Serum HCV-RNA levels were lower in complete responders than nonresponders in HCV-2a (P= .049) and HCV-2b (P= .02). The number of amino acid mutations was greater in complete responders than nonresponders in NS5A2193-2228 (the region corresponding to the ISDR of HCV-1b) alone (P= .049), or NS5A2163-2228 consisting of NS5A2193-2228 plus its upstream region (P= .02) in HCV-2a, but not in HCV-2b. A significant inverse correlation was observed between serum HCV-RNA levels and the number of amino acid mutations in NS5A2193-2228 (P= .003) or NS5A2163-2228 (P= .005) in HCV-2a. With multivariate analysis, the number of substitutions in NS5A was an independent predictor for complete response in HCV-2a (odds ratio: 6.4;P = .03). Interferon efficacy is associated with amino acid variations in the NS5A protein in HCV-2a infection.