OBJECTIVE Based on serological and molecular evidence of hepatitis C virus (HCV) infection in a significant proportion of patients with mixed cryoglobulinemia (MC), a direct association between HCV and MC has been suggested. The goal of the present study was to investigate the role played by HCV and by the immune response to the virus in the pathogenesis of mixed cryoglobulinemia. METHODS A competitive reverse transcription polymerase chain reaction was employed to evaluate the concentrations of specific HCV RNA sequences in different clinical specimens (plasma, sera, cryoprecipitates, bone marrow and peripheral blood cells). Using recombinant and synthetic peptides covering the HCV core, envelope 1 (E1) and nonstructural regions 4 (NS4) and 5 (NS5), the humoral immune response in a group of MC patients was assessed with an enzyme-linked immunosorbent assay. Natural killer (NK) cell activity was estimated using a 4 hr 51 Cr release assay. RESULTS Quantitation of the RNA molecules in the biological samples confirmed an increased virion concentration in cryoprecipitates from 13/15 patients with mixed cryoglobulinemia. Analysis of the humoral immune response against the synthetic peptides suggested a distinct response to HCV antigens in MC patients when compared to patients with HCV infection but without serological evidence of cryoglobulinemia. Unstimulated NK cell functioning was below the normal range in all patients tested. However, peripheral blood mononuclear cells showed no enhancement of NK activity by the interferon inducer polyinosinic acid:polycytidilic acid. Enhancement by interferon-alpha was normal, suggesting an impairment in interferon production. CONCLUSIONS The quantitative data are in line with the hypothesis of a direct or indirect role of HCV in mixed cryoglobulinemia. The abnormal immune response could be involved in the onset and persistence of HCV infection, and possibly in the appearance of cryoglobulinemia.