1. The effects of fluoxetine, a commonly used antidepressant drug, on G protein-activated inwardly rectifying K(+) channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays. 2. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration-dependence, a weak voltage-dependence and a slight time-dependence with a predominant effect on the instantaneous current elicited by voltage pulses and followed by slight further inhibition. Furthermore, in oocytes expressing GIRK1/2 channels and the cloned Xenopus A(1) adenosine receptor, GIRK current responses activated by the receptor were inhibited by fluoxetine. In contrast, ROMK1 and IRK1 channels in other Kir channel subfamilies were insensitive to fluoxetine. 3. The inhibitory effect on GIRK channels was not obtained by intracellularly applied fluoxetine, and not affected by extracellular pH, which changed the proportion of the uncharged to protonated fluoxetine, suggesting that fluoxetine inhibits GIRK channels from the extracellular side. 4. The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine. 5. We demonstrate that fluoxetine, at low micromolar concentrations, inhibits GIRK channels that play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate through activation of various G-protein-coupled receptors. The present results suggest that inhibition of GIRK channels by fluoxetine may contribute to some of its therapeutic effects and adverse side effects, particularly seizures in overdose, observed in clinical practice.