Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(. We found that NSC23766 inhibits the M 2 muscarinic acetylcholine receptor (M 2 mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M 2 mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rhodependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M 2 mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly G i bg-mediated, adenosine-induced activation of G i protein-regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K 1 current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A 1 receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca 21 concentrations in human embryonic kidney 293 (HEK-293) cells expressing M 1 , M 2 , or M 3 mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M 2 and M 3 mAChR crystal structures confirmed this interpretation.