We recently showed that interleukin (IL)-2-stimulated CD56+ cells derived from the liver exert vigorous cytotoxicity against hepatocellular carcinoma (HCC) by their binding to the tumor necrosis factor-related apoptosis-inducing ligand expressed on natural killer cells and the corresponding death receptors, and exhibit inhibitory effects on hepatitis C virus (HCV) replication by production of a high level of interferon-γ. These findings prompted us to develop a technique to increase the number of such innate components of cellular immunity from peripheral blood mononuclear cells (PBMCs) so that, they can be easily applied for immunotherapy clinically. We expanded CD3⁻CD56+ and CD3+CD56+ cells ex vivo from PBMCs of human volunteers by using media containing IL-2 and anti-CD3 monoclonal antibody. Among the various culture media used, autoserum supplemented X-VIVO 15 most efficiently supported PBMCs expansion and maintained the viability of the expanded cells (approximately 60-fold expansion after 28-d culture). Cultivation of PBMCs in this medium resulted in the highest proportion of CD3⁻CD56 cells among the propagated lymphocytes (approximately 40% after 28-d culture). An experiment using genomic HCV replicon-containing hepatic cells showed that the CD3⁻CD56+ cell-enriched expansion strongly inhibited HCV replication when compared with freshly isolated PBMCs. The additional anti-CD3 monoclonal antibody pulse stimulation induced anti-HCV activity even in the CD3+CD56+ cells among the propagated PBMCs. Further, cytotoxic assay showed that the expansion of CD3+CD56+ and CD3⁻CD56+ cells resulted in vigorous cytotoxicity against HCC. In conclusion, CD56+ cells obtained from the PBMCs show anti-HCV activity in addition to anti-HCC activity.