Although allosteric transitions of GroEL by MgATP 2 − have been widely studied, the initial bimolecular step of MgATP 2− binding to GroEL remains unclear. Here, we studied the equilibrium and kinetics of MgATP 2 − binding to a variant of GroEL, in which Tyr485 was replaced by tryptophan, via isothermal titration calorimetry (ITC) and stopped-flow fluorescence spectroscopy. In the absence of K + at 4–5 °C, the allosteric transitions and the subsequent ATP hydrolysis by GroEL are halted, and hence, the stopped-flow fluorescence kinetics induced by rapid mixing of MgATP 2 − and the GroEL variant solely reflected MgATP 2 − binding, which was well represented by bimolecular noncooperative binding with a binding rate constant, k on , of 9.14×10 4 M −1 s −1 and a dissociation rate constant, k off , of 14.2 s −1 , yielding a binding constant, K b (=k on /k off ), of 6.4×10 3 M −1 . We also successfully performed ITC to measure binding isotherms of MgATP 2 − to GroEL and obtained a K b of 9.5×10 3 M −1 and a binding stoichiometric number of 6.6. K b was thus in good agreement with that obtained by stopped-flow fluorescence. In the presence of 10–50 mM KCl, the fluorescence kinetics consisted of three to four phases (the first fluorescence-increasing phase, followed by one or two exponential fluorescence-decreasing phases, and the final slow fluorescence-increasing phase), and comparison of the kinetics in the absence and presence of K + clearly demonstrated that the first fluorescence-increasing phase corresponds to bimolecular MgATP 2 − binding to GroEL. The temperature dependence of the kinetics indicated that MgATP 2 − binding to GroEL was activation-controlled with an activation enthalpy as large as 14–16 kcal mol −1 .