Substitutions deduced by direct sequencing in the interferon-sensitivity-determining region (ISDR) of hepatitis C virus (HCV) are related to patients' responses to interferon (IFN), but sequencing is time consuming and results are only for the dominant virus. We developed a rapid method to detect such changes. With serum from 50 patients with chronic hepatitis C (genotype 1b) given IFN-alpha, a way to detect changes in ISDR by hybridization with oligonucleotide probes that had a prototype nucleotide sequence of HCV-J was established. Hybridization intensity was expressed as optical density (OD(NS5A)). The method was checked with serum from 100 more patients. In the study of 50 patients, all 21 with the prototype sequences had a high OD(NS5A) (> or = 0.4), and all 8 patients with a mutant-type sequence had low values (< or = 0.2). Twelve (95% confidence interval, 36-81%) of 20 patients with OD(NS5A) of <0.4 and 2 (1%-22%) of 30 patients with OD(NS5A) > or = 0.4 had complete responses (CR). All nine (66%-100%) patients with OD(NS5A) <0.4 and little HCV RNA (<100 kIU/mL) had CR, but none (0%-14%) of the 24 patients with high values from both predictors had CR. In the study of 100 patients, OD(NS5A) and the HCV RNA level were independent predictors of the effects of IFN. By multivariate analysis, the odds ratio for a CR in patients with OD(NS5A) of > or = 0.4 was 0.015 (0. 001-0.190) compared with the other patients (P =.001). In conclusion, our method should be useful in identification of prototype strains, which generally resist IFN therapy.