Three fluorescent probes TP1–3 for thiols were rationally designed and synthesized to distinguish cysteine (Cys) from glutathione (GSH)/homocysteine (Hcy). TP1–3 are almost non-fluorescent and colorless 4-nitro-1,8-naphthalimide derivatives. Upon the substitution of nitro by Cys, TP1–3 were transformed into weakly fluorescent green-emitting 4-amino analogs via highly fluorescent blue-emitting thioether intermediates. The three-channel signaling capability allows discrimination between Cys and GSH/Hcy. The fluorescence intensity at 498 nm was linearly proportional to GSH concentration in the range of 0-20 μM, and the detection limit was 5 × 10(-8) mol L(-1). A good linear relationship between A446/A350 and Cys concentration was found in the range of 0-70 μM, and the detection limit was 2 × 10(-7) mol L(-1). Moreover, TP3 was used for living cell imaging as well as for detecting mercapto-containing proteins.