The Michael addition reactions of the biothiols cysteine (Cys), glutathione (GSH), homocysteine (Hcy) and cysteinylglycine (Cys-Gly) with (E)-3-(3-(2-methoxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one and (E)-3-(3-(3-methoxyphenyl)acryloyl)-7-methoxy-2H-chromen-2-one (ChC2 and ChC3, respectively) in aqueous solution (25 degrees C, ionic strength 0.2 M KCl) were followed fluorimetrically and evaluated kinetically. With a large excess of biothiol, pseudo-first-order rate coefficients (k(obsd)) were found. Plots of k(obsd) versus [biothiolate] are linear, with a pH-independent slope (k(N)). With the kinetic results obtained, the following conclusions can be drawn: (i) the reactivity increases in the sequence Cys-Gly < Hcy < GSH < Cys; (ii) similar reactivity toward biothiols was found for ChC2 and ChC3, and both probes are more reactive than (E)-3-cinnamoyl-7-methoxy-2H-chromen-2-one (ChC1); (iii) ChC2 and ChC3 display a highly selective fluorescence enhancement with biothiols, allowing these probes to be used for fluorimetric biothiol determination in SH-SY5Y cells. Copyright (C) 2012 John Wiley & Sons, Ltd.