In vivo electroporation (EP) is an efficient method for effective gene transfer and is highly expected for application in anticancer gene therapy. Non-invasive monitoring of gene transfer/expression is critical for optimal gene therapy. Here we report in vivo optical and high-field magnetic resonance imaging (MRI) of EP-mediated transgene expression in a tumor model. Initially, we observed spatio-temporal change in in vivo EP-mediated transgene expression by optical imaging using red fluorescence protein (RFP) as a reporter gene. Next, we constructed a dual-reporter plasmid carrying a gene-encoding MRI reporter ferritin heavy chain and RFP gene to visualize the intratumoral transgene expression by dual modality. Cells transfected with this plasmid showed lower signal intensity on in vitro T-2-weighted cellular MRI and quantitatively increased the transverse relaxation rate (1/T-2) compared with control cells. After conducting in vivo EP in an experimental tumor, the plasmid-injected region showed both fluorescent emissions in optical imaging and detectably lowered signal on T-2-weighted MRI. The correlative immunohistological findings confirmed that both the reporter transgenes were co-expressed in this region. Thus, our strategy provides a platform for evaluating EP-mediated cancer gene therapy easily and safely without administering contrast agent or substrate. Gene Therapy (2009) 16, 830-839; doi:10.1038/gt.2009.55; published online 21 May 2009