A key step in advancing gene therapy protocols is to establish an efficient monitoring system following target gene delivery. Earlier, we reported our new adenoviral mediated PET reporter gene system utilizing [18F]16alpha-fluoroestradiol (FES) as PET probe and human estrogen receptor alpha ligand binding domain (hERL) as PET reporter gene and evaluated basic cell culture and initial in vivo autoradiographic studies in mice [1]. Now, we report further cell model studies and in vivo PET imaging in rats.An in vitro FES binding study was performed on MRC-5 (human lung fibroblasts) and Cos-7 (African green monkey kidney) cells infected with varying titers of test and control viruses or transfected with varying amounts of test plasmid. After 10 min uptake and 30 min intermittent wash, the FES accumulation was higher in both the cell lines infected with test virus than those transfected by test plasmid. The uptake also increased with rise in viral titer in contrast to decreasing uptake with increased plasmid amounts. The co-expression of hERL and a model therapeutic gene by the correspondingly infected cells confirmed the results of binding study.The rats were injected with test and control adenoviruses into opposite hind limb adductor muscles. In vivo PET imaging in 6-days post-infected rat yielded positive accumulation of FES on the side injected with test virus. Furthermore, the correspondingly resected muscle samples were subjected to immunohistochemical staining and the expression of reporter and therapeutic genes were confirmed. Thus, we could successfully image the expression of our reporter gene hERL in vivo in a localized area of muscle tissue in rats. We hope confirmation of reporter gene expression following infection in other target areas will enable our system to be a significant addition to the existing repertoires of reporter gene system.Joint Molecular Imaging Conferenc