Hepatitis C virus (HCV)–associated B cell lymphomas were previously shown to express a restricted repertoire of immunoglobulin V_H and V_L genes, V_H1-69 and VκA27, respectively. Although this suggests a role for antigen selection in the pathogenesis of these lymphomas, the driving antigen involved in the clonal expansion has not been identified. B cell response to a viral antigen, the HCV envelope glycoprotein 2 (E2), was analyzed in an asymptomatic HCV-infected patient. Single B cells, immortalized as hybridomas and selected for binding E2, were analyzed for their V gene usage. Sequences of these V region genes demonstrated that each hybridoma expressed unique V_H and V_Lgenes. Remarkably, these anti-E2 hybridomas preferentially used the V_H1-69 gene. Analysis of replacement to silent mutation ratios indicated that the genes underwent somatic mutation and antigenic selection. In a separate report, human anti-E2 antibodies were also shown to express the same V_H gene. These data strengthen the hypothesis that the HCV-associated lymphomas are derived from clonally expanded B cells stimulated by HCV.