Previous reports demonstrated that amino acid (aa) substitutions in the hepatitis C virus (HCV) core protein are predictors of non-virological responses to pegylated interferon (Peg-IFN) and ribavirin combination therapy. The aim of this study was to investigate the impact of core aa substitutions on viral kinetics during the treatment and relapse after the treatment. The 187 patients with HCV genotype 1 enrolled in this study were categorized into four groups according to core aa substitution patterns: double-wild group (n = 92), Arg70/Leu91; 70-mutant group (n = 42), Gln70/Leu91; 91-mutant group (n = 31), Arg70/Met91; and double-mutant group (n = 22), Gln70/Met91. The relationship between the core aa substitutions and the virological response was examined. Multivariate logistic regression analyses showed that substitution at aa 70 was significantly associated with a poor virological response during the first 12 weeks (decline of <1 log from baseline at week 4, <2 log at week 12), and substitution at aa 91 was significantly associated with detectable HCV RNA at week 24. With respect to relapse, only the ribavirin exposure (odds ratio (OR), 0.77; 95% confidence interval (CI), 0.60–0.98) and HCV RNA disappearance between weeks 13 and 24 (OR, 23.69; 95% CI, 5.44–103.08) were associated independently with relapse, with no correlation being found with the core aa substitutions and relapse. In conclusion, the results showed that core aa substitutions can be strong predictive factors at pretreatment of the non-response, but not for relapse, for virological responders with HCV RNA disappearance during treatment. J. Med. Virol. 83:419–427, 2011. © 2011 Wiley-Liss, Inc.