Guanine nucleotides and Na+ are known to regulate ligand binding to cardiac muscarinic receptors, which are negatively coupled to the adenylate cyclase system. In the present study, we found that NH+4 was more potent than Na+ or other monovalent cations in regulating the affinity of the muscarinic receptor for agonists and antagonists. The effect of NH+4 (or Na+) on the binding of the antagonist [3H]quinuclidinyl benzilate (QNB) to muscarinic receptors in homogenates of embryonic chick hearts depended on the assay buffer used. NH+4 increased Kd in phosphate buffer or histidine and increased Bmax in Tris. NH+4 (0.1 M) increased the IC50 value for acetylcholine inhibition of [3H]QNB binding 20-fold compared to 3-4-fold with 0.1 M Na+ or K+. Furthermore, NH+4 could substitute for and was more potent than Na+ in producing synergistic effects with Gpp[NH]p to reduce the affinity of the receptor for acetylcholine. Tris depressed these effects. Gpp[NH]p plus 0.4 M NH4Cl totally converted the receptor population to a low affinity agonist state and increased the IC50 for acetylcholine by more than 2000-fold. Two conclusions can be made from the present results. First, NH+4 appears to be the most potent effector yet studied of the monovalent cation site of the muscarinic receptor system. Second, the use of Tris in muscarinic receptor ligand binding assays will produce anomalous results concerning the properties of both agonist and antagonist binding to the receptor.