We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGluh, iGlum, and iGlul cover a broad range of Kd-s (5.8 μM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a Kd of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGluu and iGluf had monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlum and iGlul, the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGluh and Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion-limited rate constant to iGluh and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding.