Angiotensin-converting enzyme 2 (ACE2) is the main molecular target for coronavirus SARS-CoV-2 to enter cells. Molecularly specific tracers that bind to ACE2 with high affinity can be used to determine the tissue distribution of this important receptor, noninvasively. A novel targeting PET imaging probe, [18F]AlF-DX600-BCH, was developed to detect the in vivo expression of ACE2 and monitor response to therapy. Preclinical experiments, including biodistribution, PET imaging, and tissue section analysis, were conducted after tests of in vitro and in vivo stability and pharmacokinetics. The agent was advanced to clinical evaluation in 10 volunteers who received [18F]AlF-DX600-BCH PET/CT at 1 and 2 h after injection (NCT04542863). Preclinical results of both biodistribution and PET demonstrated [18F]AlF-DX600-BCH accumulation in rat kidney (standardized uptake value; SUVkidney/normal > 50), along with specific uptake in testes (SUVtestis/normal > 10) tissues. Kidney, gastrointestinal, and bronchial cell labeling were correlated to ACE2 positive by immunohistochemistry (IHC) staining. In clinical imaging, significant tracer accumulation was predominantly observed in the urinary and reproductive system (SUVrenal cortex = 32.00, SUVtestis = 4.56), and the conjunctiva and nasal mucosa saw elevated uptake in several cases. This work is the first report of a radioisotope probe, [18F]AlF-DX600-BCH, targeting ACE2 with promising preliminary preclinical and translational outlook, thereby demonstrating the potential of noninvasive mapping of ACE2.© 2024 American Chemical Society.