We investigated the pathogenetic relevance of hepatitis C virus (HCV) infection in mixed cryoglobulinemia (MC) with or without complicating B-cell Non-Hodgkin's lymphoma (NHL) in comparison with other immunological and lymphoproliferative disorders. The following groups of patients were studied: A) 25 patients with MC in 7 cases evolved into B-cell NHL; B) 25 healthy subjects; C) 22 patients with different systemic immune diseases; D) 24 patients with chronic HCV infection without MC; E) 25 patients with B-cell idiopathic NHL. Methods used included: i) Polymerase chain reaction (PCR) for HCV RNA detection in serum and peripheral blood mononuclear cells (PBMC) (uncultured or mitogen-stimulated); ii) Branched DNA (b-DNA) for HCV RNA quantification; iii) HCV genotyping by genotype-specific primers localized in the core region and by hybridization of amplification products of the 5' untranslated region (5'UTR), obtained with universal primers, using genotype-specific probes. Serum anti-HCV and HCV RNA were detected in 88% and 73% of MC patients, respectively, and in a significantly lower percentage of healthy controls and patients with autoimmune diseases. HCV RNA concentration was significantly lower in supernatants than in corresponding whole sera (p < 0.001). Plus-strand HCV RNA was detected in 81% of peripheral blood mononuclear cell (PBMC) samples and minus-strand in the majority of fresh or mitogen stimulated cells. All MC patients with NHL had HCV RNA sequences in PBMC. HCV genotype 2a/III was detected in MC patients with a prevalence that was significantly higher than in HCV infected patients without MC. Surprisingly, HCV markers (anti-HCV and/or HCV RNA) were found in 32% of patients with idiopathic NHL. These data suggest that HCV infection is involved in the pathogenesis of MC through both direct participation in the immune complex related vasculitis and by triggering the lymphoproliferative disorder underlying the disease. This latter disorder seems to be related to HCV lymphotropism which could also be responsible for the evolution of MC to malignant lymphoma. This study also suggests that HCV infection may be involved in the pathogenesis of idiopathic B-cell NHL through a similar pathogenetic mechanism.