Methylene Blue (MB) is being investigated in clinical studies for its beneficial effects in the treatment of Alzheimer disease. However, its exact mechanisms of action have not been fully elucidated. The modulation of nicotinic acetylcholine receptors (nAChRs) has been suggested to play a role in the pathogenesis of various neurodegenerative diseases. Therefore, in the present study, the effect of MB on the function of the cloned α7 subunit of the human nAChR expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. MB reversibly inhibited ACh (100 μM)-induced currents in a concentration-dependent manner with an IC50 value of 3.4 ± 0.3 μM. The effect of MB was not dependent on the membrane potential. MB did not affect the activity of endogenous Ca2+-dependent Cl- channels, since the inhibition by MB was unaltered in oocytes injected with the Ca2+ chelator 1,2-bis (o-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid and perfused with Ca2+-free bathing solution containing 1.8 mM Ba2+. MB decreased the maximal ACh-induced responses without significantly affecting ACh potency. Furthermore, specific binding of [125I] α-bungarotoxin, a radioligand selective for the α7 nAChR, was not altered by MB (10 μM), indicating that MB acts as a noncompetitive antagonist on α7 nAChRs. In hippocampal slices, whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the γ-aminobutyric acid-mediated spontaneous postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for α7 nAChRs, were abolished after 10 min application of 3 μM MB. These results demonstrate that MB inhibits the function of human α7 nAChRs expressed in Xenopus oocytes and of α7 nAChR-mediated responses in rat hippocampal neurons.