Both a double-stranded RNA-dependent protein kinase (PKR)-phosphorylation homology domain (PePHD) within the E2 protein and a PKR-binding domain within the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 isolates inhibit the function of the interferon alfa (IFN-α)-induced antiviral effector protein PKR in vitro. We investigated whether the mutational pattern of the E2 region (codons 618-681, including PePHD) of 81 HCV genotype 1-infected patients (HCV-1b [n = 54], HCV-1a [n = 27]) influences the response to IFN-α. Initial viral decline (ΔHCV RNA) was determined at week 1 hereby covering the effector reactions of IFN-α-mediated first phase and the immune-mediated second phase. ΔHCV RNA less than 50% (group 1); ΔHCV RNA greater than 50% but less than 90% (group 2); and ΔHCV RNA ≥90% (group 3) were differentiated. The PePHD region was highly conserved; the few mutations (5 patients) did not correlate with ΔHCV RNA or sustained virologic response to IFN-α. Within the flanking regions before and after PePHD (codons 618-681) 72 of 81 patients (89%) had 2.6 ± 0.17 mutations (median, 3; range, 1-8) that did not correlate with treatment response. Sequence analysis of the NS5A protein (codons 2,209-2,274, including interferon sensitivity determining region [ISDR]) in 39 of 81 patients showed a higher mean number of mutations in the ISDR (codons 2,209-2,248) in groups 2 (1.28 ± 0.43 [n = 18]) and 3 (1.89 ± 0.54 [n = 9]) than in group 1 (0.67 ± 0.19 [n = 12]; P = .049 group 1 vs. 3) and a mutant type ISDR (e.g., ≥4 mutations) was significantly more frequent in sustained virologic responders than in nonresponders or relapsers (2 of 4 [50%] vs. 2 of 35 [6%]; P = .045). Thus, NS5A appears to be functionally relevant in IFN-α-induced effector reactions.