Agonist activation of muscarinic acetylcholine (mACh) receptors in porcine atrial membranes stimulates binding of the GTP analog, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins. In contrast, atropine as well as several other mACh receptor antagonists reduced the binding of GTP[S] below basal values, both in the absence and presence of the agonist carbachol, by a similar maximal extent (about 25%). Evidence is presented that this inhibitory action of atropine was not due to an antagonism of endogenous acetylcholine. Similar to agonist-induced stimulation, antagonist-induced inhibition of GTP[S] binding required the presence of GDP and Mg2+. On the other hand, addition of salts, e.g. NaCl, amplified agonist but reduced antagonist effects on GTP[S] binding. The data presented suggest that agonist-unliganded mACh receptors interact with and activate G proteins in native cardiac membranes and that antagonist binding induces a conformational change of the receptor, which then either does not interact with G proteins or which prevents GDP release from and subsequent GTP[S] binding to G proteins.