A high prevalence of hepatitis C virus (HCV) genotype 2c (22%) was detected in sera from 459 italian patients by core-region amplification and hybridization with specific probes by DNA enzyme immunoassay. Amplified fragments failed to hybridize with 1a, 1b, 2a, 2b and 3a subtype-specific and 4, 5, 6 type-specific oligonucleotides in 105 patients. Hybridization of these samples with type 2 probe, which recognized all the subtypes sequences, showed evidence for genotype 2 distinct from 2a and 2b. Fourteen out of these 105 isolates were cloned and sequenced. The results were consistent with genotyping assay. Nucleotide sequences were partially related to types 2a, 2b, 2d, 2e and 2f (87.0-93.5% of identity). The average nucleotide identity was highest for genotype 2c (95.87%). On the basis of sequence analysis, subtype 2c specific probe was derived. Hybridization efficiency with the newly designed probe was very high and more than 95% (100/105) of type 2 cases were classified as 2c. Evidence of different outcome of therapy inside the same HCV major type account for the need of accurate subtyping. In this study, amplification of the core region followed by hybridization with highly specific probes enabled distinction between HCV subtypes.