In cellular signaling, adenylyl cyclase plays a key role in the hydrolysis of ATP to cyclic AMP and pyrophosphate. Using a synthetic fluorescent chemosensor (PyDPA) which binds strongly to the pyrophosphate group, we have developed a label-free fluorescent real-time detection system for adenylyl cyclase. This assay would be the first adenylyl cyclase assay based on chemosensing the production of pyrophosphate. (C) 2009 Elsevier Ltd. All rights reserved