Hepatitis C virus (HCV) is a major worldwide cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The development of vaccines against HCV have been complicated by the high variability of the envelope region, and it is likely that the cellular immune responses to viral structural proteins may be important for eradicating persistent viral infection. Recently, it was reported that the injection into muscle cells of plasmids encoding viral genes resulted in the generation of strong cellular immune responses. We constructed vectors that express the highly conserved HCV core gene. In this regard, the pHCV 2-2 construct contained the entire HCV core region and pHCV 4-2 contained both the 5' noncoding region and the core gene. Cellular expression of HCV core protein was assessed following transfection into human and murine cell lines, and higher intracellular levels of the 21-kd core protein were observed with pHCV 2-2. These HCV core DNA constructs were used to immunize BALB/c mice and produced low-level anti-HCV core humoral immune responses. To assess cytotoxic T-lymphocyte (CTL) activity generated in vivo, a cloned syngeneic SP2/O myeloma cell line constitutively expressing HCV core protein was established and inoculated into BALB/c mice to produce growth of plasmacytomas. Strong CTL activity was generated because the tumor size and weight in pHCV 2-2-immunized mice were remarkably reduced compared with mice injected with mock DNA. Spontaneous CTL activity was also exhibited by splenocytes in an in vitro cytotoxicity assay. These investigations demonstrate that plasmid constructs expressing HCV core protein generate strong CTL activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents.