A series of coumarin fluorophores (1-3), each bearing a double bond conjugated quinoline unit that can undergo a Michael-type reaction with thiol-containing compounds, is reported. These systems, designed to provide so-called turn-on changes in fluorescence response when exposed to thiols, act as fluorescent chemical sensors for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). In the case of 1, selectivity for Cys over Hcy and GSH is observed, both in terms of analyte-induced signal enhancement and response time. On the basis of fluorescence spectroscopic analyses, DFT calculations, and pH dependent studies this substrate selectivity is ascribed to steric interactions between the substituents on the quinolone units present in 1 and the targeted thiols, as well as to the comparatively lower pK(a) value of Cys relative to Hcy and GSH. In aqueous solution, probe 1 was found capable of detecting Cys with a detection limit of 10(-7) m. This system was successfully applied to the fluorescence imaging of intracellular Cys in HepG2 cells.