Fluoxetine is used in the treatment of a variety of clinical disorders including depression and obesity, and of cocaine detoxification or alcoholism. It is generally believed that fluoxetine exerts its clinical effects because it selectively blocks 5-hydroxytryptamine (5HT) reuptake into nerve terminals. In here we describe that fluoxetine antagonized the neuronal homomeric α7 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes, with an IC50 of 43 μM, when fluoxetine was coapplied with ACh, and of 1.6 μM when the oocytes were pretreated briefly with fluoxetine. A similar block occurred in oocytes expressing L247T α7 mutant nAChR. Furthermore, blockage of mutant α7 receptors appeared non-competitive and was stronger with cell membrane hyperpolarization. Cell-attached single channel recordings in oocytes expressing L247T α7 mutant nAChR showed that the voltage-dependence of the blockage by fluoxetine could be due to a drastic decrease in channel opening frequency accompanied by marked channel flickering and reduced channel conductance. We conclude that fluoxetine behaves as a reversible blocker of both wild and mutant α7 receptors; and that the Leu-247T mutation in the channel domain renders the blockage of α7 nAChR by fluoxetine voltage-dependent. These effects of fluoxetine on α7 receptors may be clinically important.