Hepatitis C virus (HCV) genotype 1b and high pretreatment virus load are predictive factors of poor response to interferon therapy in patients with chronic hepatitis C. To further examine the factors predicting the response to interferon in patients with genotype 1b infection, we analyzed 110 consecutive patients with HCV who were treated with a total of 624 million units of lymphoblastoid interferon alfa. Thirty-six patients (33%) were responders, while the remaining 74 patients (67%) were nonresponders. Multivariate analysis showed that a high virus titer (assessed by serum core protein level, P = .0021) and the presence of more than two amino acid substitutions in the interferon sensitivity-determining region (ISDR) (P = .0036) correlated significantly with the response to interferon therapy. Because mutations analyzed by direct sequencing of polymerase chain reaction (PCR) products may reflect artifacts of direct sequencing, we further analyzed quasispecies of HCV in this region by cloning and sequencing. Although PCR-based analysis of responders with multiple amino acid substitutions in the ISDR showed the presence of a small amount of wild-type strain in their serum, the results obtained by direct sequencing and cloning were essentially the same. A longitudinal study of quasispecies in 2 patients who showed a dramatic change in the virus titer showed no conversion from wild type to mutant or vice versa. Our results indicate that amino acid substitutions and virus load are independent predictors of the response to interferon therapy. The ability of some patients with no mutation in the ISDR or high virus load to eliminate the virus suggests the presence of other unidentified factors, host or viral, that influence the response to interferon therapy.