Purpose We identified and validated 18 F-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells. Procedures CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro . The biodistribution and metabolism pattern of 18 F-CP18 were assessed in vivo . 18 F-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity. Results In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo , 18 F-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of 18 F-CP18 retention in the dexamethasone-induced thymus of treated versus control mice. Conclusions We report the use 18 F-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.