Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.