Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.