In a vesicle preparation from rat cerebral cortex, carbachol recognizes a high-affinity and a low-affinity muscarinic agonist binding site. A number of agents, including veratridine (10 microM), gramicidin (10 microM) and valinomycin (10 microM), which depolarize the vesicles also appear to block the high-affinity muscarinic agonist binding site. Lowering [Na+] (from 137 to 80 mM) or raising [K+] (from 5 to 50 mM) produced effects similar to those of the depolarizing agents. Agents such as tetrodotoxin (1 microM), which block the Na+-channel, also appear to block the high-affinity agonist binding site or to convert it into a low-affinity agonist binding form.