Autoinducer-2 (AI-2) is a quorum sensing signal that mediates communication within and between many bacterial species. However, its known receptors (LuxP and LsrB families) are not found in all the bacteria capable of responding to this signaling molecule. Here, we identify a third type of AI-2 receptor, consisting of a dCACHE domain. AI-2 binds to the dCACHE domain of chemoreceptors PctA and TlpQ of Pseudomonas aeruginosa, thus inducing chemotaxis and biofilm formation. Boron-free AI-2 is the preferred ligand for PctA and TlpQ. AI-2 also binds to the dCACHE domains of histidine kinase KinD from Bacillus subtilis and diguanylate cyclase rpHK1S-Z16 from Rhodopseudomonas palustris, enhancing their enzymatic activities. dCACHE domains (especially those belonging to a subfamily that includes the AI-2 receptors identified in the present work) are present in a large number of bacterial and archaeal proteins. Our results support the idea that AI-2 serves as a widely used signaling molecule in the coordination of cell behavior among prokaryotic species.