Fluoxetine is a selective serotonin reuptake inhibitor used widely in the treatment of depression. In contrast to the proconvulsant effect of many antidepressants, fluoxetine has anticonvulsant activity. This property may be due in part to positive modulation of the GABA(A) receptors (GABARs), which mediate most fast inhibitory neurotransmission in the mammalian brain. We examined the effect of fluoxetine on the activity of recombinant GABARs transiently expressed in mammalian cells. Fluoxetine increased the response of the receptor to submaximal GABA concentrations but did not alter the maximum current amplitude. Sensitivity did not depend upon the beta- or gamma-subtype composition of the receptor when coexpressed with the alpha(1) subunit. Among the six alpha subtypes, only the alpha(5) subunit conferred reduced sensitivity to fluoxetine. The metabolite norfluoxetine was even more potent than fluoxetine. Mutations at residues in the alpha(5) subunit that alter its sensitivity to zinc or selective benzodiazepine derivatives did not affect potentiation by fluoxetine. This suggests that fluoxetine acts through a novel modulatory site on the GABAR. The direct positive modulation of GABARs by fluoxetine may be a factor in its anticonvulsant activity.