The RNAs of hepatitis C virus (HCV) isolates from 62 patients with chronic HCV infection were analyzed by direct sequencing of the 5' untranslated region. Two important sequence motifs were recognized: one between positions -170 and -155 and the other between positions -132 and -117. These motifs are partly complementary. All three previously published genotypes were observed; 34 (55%) isolates were classified as type 1 (including prototype [from the United States] and HCV-BK [from Japan] sequences), 11 (18%) were classified as type 2 (including HC-J6 and HC-J8), and 12 (19%) were classified as type 3 (including EB1); one patient was infected with genotypes 1 and 2. Four (6%) isolates showed aberrant sequences and were therefore provisionally classified as genotype 4. These results indicate the significance of sequence variation among the 5' untranslated regions of different HCV genotypes and indicate that this region could possibly be used for consistent genotyping of HCV isolates.