Hypervariable region I (HVRI) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) undergoes sequential alterations at intervals of several months during the chronic phase of hepatitis. To evaluate the implications of sequence variability in HVRI of HCV, we investigated the sequence variability of the whole envelope-protein(gp35 and gp70)-coding regions of HCV genome derived from patient M in acute and relapsed phases (8-month interval) of hepatitis. From this analysis, we found that a Leu (position 405) in HVRI substituted to Pro, and that 4 additional substitutions could be detected in gp70 during the relapsed phase. Sequence-specific antibody against HVRI derived from patient M was first detected in the serum at 8 months after the onset of hepatitis, but no other specific antibodies against peptides containing amino-acid position(s) substituted in regions other than HVRI could be detected. Epitope mapping using the sequence of HVRI derived from the acute phase of hepatitis was also performed, and a B-cell epitope (positions 397 to 407) of 11 amino acids was identified. However, the Pro variant at position 405 did not display an escape pattern from the antibody produced at 8 months after the onset. In addition, we demonstrated the existence of important amino-acid residue positions which are recognized by the anti-HVRI antibody produced in patient M using introduction point mutations within HVRI.