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We recently characterized LY2033298 as a novel allosteric modulator and agonist at M4 muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M4 mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M4 mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [35S]-GTPγS binding, or effects on equilibrium binding of [3H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.LY2033298 had similar affinities for the human and mouse M4 mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M4 mAChR. At the mouse M4 mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M4 mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M4 mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to ‘probe dependence’. This has implications for the validation of allosteric modulator actions in vivo.