Real-time imaging of gene expression in vivo at high spatial resolution would significantly aid our ability to study and understand transgene expression in live animals and potentially in a clinical gene therapy setting. Magnetic resonance (MR) imaging has recently achieved spectacular image resolution (50 μm3 voxel resolution in vivo), making possible imaging at very high resolutions in small animals, during development and in clinical practice. Recently we have developed gene-targeted MR imaging contrast agents that allow us to use clinical MR imaging systems to monitor gene expression in vivo. We have demonstrated that expression of an altered form of the transferrin receptor (ETR) can be imaged in vivo using MR imaging and ETR-targeted imaging probes1. ETR expression could be used as a surrogate marker to monitor viral delivery and expression of other transgenes in real time, in vivo and potentially in a clinical setting. To demonstrate the feasibility of this approach we have constructed amplicons, based on the herpes simplex virus, that co-express the ETR and different therapeutic or marker genes under the control of different constitutive promoters. In vitro we have shown that these amplicons drive the expression of multiple inserted genes and that the expression of the ETR is correlated with the expression of other genes contained within the same amplicon. Using these amplicons in a mouse xenograft brain tumor model we have demonstrated non-invasive MR imaging of in vivo transferred gene products. The results demonstrate the feasibility and power of this new approach; identification of targets overexpressed in different disease states will expand the potential application of this technology as a noninvasive diagnostic tool.