Hepatitis C virus (HCV) isolates, obtained from 50 Italian patients with community-acquired infection, that had previously been classified as subtype 2a or 2b by current rapid genotyping methods were further characterized by partial sequence analysis. All the isolates were reclassified: 45 within subtype 2c and the other 5 as subtype 1b, 3a, or 4d. Thus, subtype 2c is much more prevalent than previously recognized, with about 30% of all HCV strains detected in Italy being subtype 2c. In contrast, isolates of subtypes 2a and 2b appear to be infrequent, if not absent. Further studies showed that subtype 2c isolates are heterogeneous in the NS5A region, in that they may or may not contain a 57-nucleotide (nt) segment spanning from nt 7533 to nt 7589 of the viral genome. Partial nucleotide sequencing of the NS5B region of four 2c subtypes excluded the possibility that the isolates possessing or not possessing the 57-nt segment in the NS5A region may have resulted from recombination phenomena.