Essential mixed cryoglobulinemia is frequently associated with hepatitis C virus (HCV) infection, with the formation of HCV antigen/antibody complexes. The hypervariable region (HVR) of the HCV E2/NS1 region is thought to include epitopes for neutralizing antibodies, but it remains uncertain whether cryoglobulins (CGs) contain such antibody-bound HCV. Thus, we studied HVR clones isolated from cryoprecipitate and supernatant in the sera of four chronic hepatitis C patients with cryoglobulinemia, and expressed as fusion proteins with glutathione S-transferase (GST). Patients' sera were tested for antibody binding to the proteins. The rate of anti-HVR antibody-positive clones was significantly higher in cryoprecipitate (89% ± 13%, P < .05) than in supernatant (41% ± 25%). Both HCV RNA and anti-HVR antibody were more concentrated in cryoprecipitates compared with those of serum and supernatant in two patients tested. Anti-HVR antibody-positive clones in cryoprecipitate showed common amino acid (aa) sequences in each of the four patients. Similarly, all the antibody-positive clones in supernatant showed the same aa sequences for three of the four patients. When aa sequences were compared with those of reported isolates with genotype 1b, the mean percentage of aa difference was greater in the clones from supernatant and in anti-HVR antibody-negative clones than in the clones from cryoprecipitate and in the antibody-positive clones, respectively. These findings indicate that serum CG contains anti-HVR antibody-bound HCV in patients with chronic hepatitis C. Anti-HVR antibody-free individual clones, which were more frequently noted in supernatant, showed closely related sequences, but which were of a heterogeneous quasispecies nature.