The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT(4) receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT(4) receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT(4) receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5-10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT(4) receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT(4) receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.