Phencyclidine (PCP) is a non-competitive inhibitor of the nicotinic acetylcholine receptor (nAChR) with biphasic characteristics. At low and high micromolar concentrations, PCP inhibits nAChR from fetal mouse muscle, whereas at intermediate concentrations PCP does not inhibit the receptor. The present study was performed to determine whether the high and low concentration effects of PCP on mouse nAChR were due to interactions of this blocker with channel lining amino acids. In order to test this hypothesis, we examined the ability of PCP to inhibit acetylcholine-induced currents from wild-type nAChR and nAChR in which amino acid substitutions were made in the 6Ј, 8Ј and 10Ј positions of the M2 transmembrane segments of the receptor. Fetal mouse nAChR from BC 3 H-1 cells were expressed in Xenopus laevis oocytes and studied using the two-electrode voltage clamp technique. The results of this study reveal that in native fetal muscle receptor, PCP potency is not affected by membrane potential between Ϫ80 mV and Ϫ30 mV. The potency of PCP is increased by mutations in M2 6Ј, 8Ј, and 10Ј positions. This increase in potency cannot be explained merely by either changes in hydrophobicity/hydrophilicity of amino acids at these positions or by side-chain size. A model proposing extraluminal inhibitory and regulatory sites for PCP explains the lack of voltage-dependency, the biphasic effect of PCP, and the fact that all M2 mutations increased PCP potency (by disrupting the link with the regulatory sites).