Positron emission tomography (PET) reporter systems are a valuable means of estimating the level of expression of a transgene in vivo. For example, the safety and efficacy of gene therapy approaches for the treatment of neurological and neuropsychiatric disorders could be enhanced via the monitoring of exogenous gene expression levels in the brain. The present study evaluated the ability of a newly developed PET reporter system 18Ffluoroestradiol (18FFES) and the estrogen receptor-based PET reporter ChRERα, to monitor expression levels of a small hairpin RNA (shRNA) designed to suppress choline acetyltransferase (ChAT) expression in rhesus monkey brain. The ChRERα gene and shRNA were expressed from the same transcript via lentivirus injected into monkey striatum. In two monkeys that received injections of viral vector, 18FFES binding increased by 70% and 86% at the target sites compared with pre-injection, demonstrating that ChRERα expression could be visualized in vivo with PET imaging. Post-mortem immunohistochemistry confirmed that ChAT expression was significantly suppressed in regions in which 18FFES uptake was increased. The consistency between PET imaging and immunohistochemical results suggests that 18FFES and ChRERα can serve as a PET reporter system in rhesus monkey brain for in vivo evaluation of the expression of potential therapeutic agents, such as shRNAs. Display omitted Eldridge, Michaelides, and colleagues successfully applied 18FFES-ChRERα in rhesus monkeys, which increases the likelihood that this new PET reporter system will translate effectively to the human brain. PET reporter systems could be valuable for gene therapy-based treatments of neuropsychiatric and neurological disorders.