Muscarinic receptors trigger several different responses including an increase in concentration of cyclic GMP, a decrease in cyclic AMP concentration, breakdown of polyphosphoinositides and changes in ion permeability. It is not yet clear whether these reactions occur sequentially or independently and which directly coupled to the muscarinic receptor. Several lines of evidence indicate that muscarinic receptors in many, if not all, cell types are coupled to the inhibitory guanine nucleotide regulatory protein (Ni or Gi) of adenylate cyclase. To provide direct evidence for this coupling, we have reconstituted muscarinic receptors purified from porcine brain with Ni purified from rat brain in a phospholipid vesicle. Here, we report that the GTPase activity of Ni is stimulated by carbachol. This action is blocked by the simultaneous addition of atropine and is not observed when the Ni protein is ADP-ribosylated. We conclude that one function of the muscarinic receptor is the activation of Ni.