Bacterial histidine kinases transduce extracellular signals into the cytoplasm. Most stimuli are chemically undefined; therefore, despite intensive study, signal recognition mechanisms remain mysterious. We exploit the fact that quorum-sensing signals are known molecules to identify mutants in the Vibrio cholerae quorum-sensing receptor CqsS that display altered responses to natural and synthetic ligands. Using this chemical-genetics approach, we assign particular amino acids of the CqsS sensor to particular roles in recognition of the native ligand, CAI-1 (S-3 hydroxytridecan-4-one) as well as ligand analogues. Amino acids W104 and S107 dictate receptor preference for the carbon-3 moiety. Residues F162 and C170 specify ligand head size and tail length, respectively. By combining mutations, we can build CqsS receptors responsive to ligand analogues altered at both the head and tail. We suggest that rationally designed ligands can be employed to study, and ultimately to control, histidine kinase activity.