We recently described 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298) as a novel allosteric modulator of M(4) muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M(4) mAChR subtype. In the current study, we show that LY2033298 can also bind to the M(2) mAChR and mediate robust positive or negative allosteric effects, depending on the orthosteric ligand used as a probe of receptor activity. This finding of striking probe dependence indicates that the previously described selectivity of the modulator does not arise as a consequence of selective affinity for a poorly conserved allosteric site but rather is due to subtype-selective cooperativity with ACh upon interaction with a common allosteric binding site. Moreover, a comparison of the effects of the modulator on orthosteric ligand affinity relative to signaling through a [(35)S]guanosine 5 -O-(3-thio)triphosphate or extracellular signal-regulated kinase 1/2 phosphorylation assay at the M(2) mAChR revealed that, although the effects on binding were positive in all instances, the effects on signaling were either positive or strongly negative, depending on the agonist and the pathway. Mutational analysis identified residues Tyr177 and Trp99(3.28) (Ballesteros and Weinstein numbers are provided in superscript to indicate relative position of residues within the transmembrane domain) as contributing to the binding of LY2033298, whereas the orthosteric site residues, Tyr104(3.33) and Tyr403(6.51), contributed to the ability of the ligand to impose pathway-biased modulation. Taken together, these findings have important implications for the detection and validation of allosteric modulators of G protein-coupled receptors (GPCRs), because they highlight the potential for ligand misclassification or lack of appreciation of off-target allosteric activities.