While many orthosteric ligands have been developed for the mGlu 2 receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e. k on and k off , of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric mod-ulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay. We found that dissociation rate constants of orthosteric mGlu 2 ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu 2 ligands is k on-driven in vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on-and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency. In conclusion, we found that affinity-or potency-based orthosteric ligand optimization primarily results in ligands with high k on values. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasing k off , which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacy in vivo.