The effects of the antipsychotic clozapine on the function of the cloned α7 subunit of the nicotinic acetylcholine (nACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Clozapine reversibly inhibited nicotine (10 μM)-induced currents in a concentration-dependent manner (300 nM to 90 μM), with an IC50 value of 3.2 ± 0.4 μM. The effect of clozapine was not dependent on the membrane potential. Clozapine did not affect the activity of endogenous Ca2+-dependent Cl− channels since the inhibition by clozapine was unaltered by the intracellularly injected Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Clozapine decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on α7-nACh receptors. In hippocampal slices, the whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the GABA-mediated spontaneous inhibitory postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for α7-nACh receptors, were abolished after 10 min application of 5 μM clozapine. In conclusion, these results demonstrate that clozapine inhibits the function of α7-nACh receptors expressed in Xenopus oocytes and in hippocampal neurons.