Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the G(q/11) family and m2 and m4 to members of the G(i/0) family. We have recently shown that Drosophila melanogaster and other arthropods have two mAChRs, named A and B, where the A-type has the same pharmacology as the mammalian mAChRs, while the B-type has a very low affinity to muscarine and no affinity to classical antagonists such as atropine. Here, we find that the D. melanogaster A-type mAChR is coupled to G(q/11) and D. melanogaster B-type mAChR to G(i/0). Furthermore, by comparing the second and third intracellular loops of all animal mAChRs for which the G protein coupling has been established, we could identify several amino acid residues likely to be specific for either G(q/11) or G(i/0) coupling. Using these hallmarks for specific mAChR G protein interaction we found that all protostomes with a sequenced genome have one mAChR coupled to G(q/11) and one to four mAChRs coupled to G(i/0). Furthermore, in protostomes, probably all A-type mAChRs are coupled to G(q/11) and all B-type mAChRs to G(0/i) (C) 2015 Elsevier Inc. All rights reserved.