Background and Purpose We recently characterized LY2033298 as a novel allosteric modulator and agonist at M(4) mAChRs. Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M(4) mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalise its in vivo actions. Experimental Approach LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M(4) mAChRs, using assays of agonist-induced ERK1/2 or GSK3alpha phosphorylation, [(35) S]-GTPgammaS binding, or effects on equilibrium binding of [(3) H]-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. Key Results LY2033298 had similar affinities for the human and mouse M(4) mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M(4) mAChR. At the mouse M(4) mAChR, LY2033298 showed higher cooperativity with oxotremorine than with ACh or xanomeline. The different degrees of cooperativity between LY2033298 and each agonist at the mouse relative to the human M(4) mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298. Conclusions and Implications These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M(4) 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.