The pH-dependence of antagonist and agonist binding to rat heart muscarinic receptors was investigated at 25 degrees C, in the absence and in the presence of GTP. The small inhibitory effect observed at the lowest pH investigated (pH 6.0) on [N-methyl-3H]methscopolamine chloride and [methyl-3H]oxotremorine-M acetate binding indicated that one or more amino acid residues of the receptor had to be deprotonated for optimal binding affinity. The low pK value of these residues (between 5 and 6) prevented their identification. The binding of scopolamine (pK 7.6) was favoured by a positive charge in the titratable amine, but binding with a lower affinity remained possible charge in the titratable amine, but binding with a lower affinity remained possible without this charge. GTP did not affect antagonist binding at any pH, but converted more than 90% of agonist binding sites into a low affinity conformation. In the absence of GTP, we observed a time- and pH-dependent conversion of the super-high- and high-affinity receptors to a low-affinity GTP-insensitive state. This conversion was markedly accelerated at high pH (above pH 8.0). In the presence of GTP, a positive charge on the titratable amine of pilocarpine (pK 7.05) and oxotremorine (pK 8.60) was required for binding. These results support the view that antagonist (e.g. methscopolamine) binding to receptors was largely facilitated by hydrophobic interactions, whereas agonist binding to low-affinity sites was mainly driven by ionic interactions.