Tris and choline reduce the maximal binding capacity (RT) of the muscarinic cholinergic antagonist [3H]-L-quinuclidinyl benzilate ([3H]-L-QNB) to atrial membranes, when compared to control values in physiological salt solution (PBS) or NaPi buffer. Addition of guanine nucleotides (GN) to incubations containing choline or Tris reverses the effect of choline and Tris on RT and restores it to levels determined in NaPi or PBS alone. GN addition fails to alter RT or KD values determined in NaPi or PBS in the absence of choline and Tris. This GN effect follows a nucleotide specificity similar to that of the GN regulatory proteins coupled to adenylate cyclase. Tris or choline are required for the expression of GN regulation of [3H]-L-QNB binding to muscarinic acetylcholine receptors (mAChR). An allosteric site recognizing choline and Tris appears involved in the interaction between the guanine nucleotide regulatory protein and antagonist binding to mAChR.