In vivo extracellular unitary recordings, in vitro 3H-5-hydroxy- tryptamine (5-HT) uptake, and 3H-paroxetine binding assays were used to assess the effect of acute and long-term administration of the 5-HT reuptake inhibitor paroxetine on the neuronal 5-HT transporter in the rat dorsal hippocampus. Recovery time of the firing activity of CA3 hippocampus pyramidal neurons following microiontophoretic application of 5-HT was used as an index of in vivo reuptake activity. In a first series of experiments, the acute intravenous administration of paroxetine and 5-HT denervation with the neurotoxin 5,7- dihydroxytryptamine produced a marked prolongation of the suppressant effect of 5-HT, indicating that reuptake into 5-HT terminals plays a significant role in terminating the action of microiontophoretically applied 5-HT. In a second series of experiments, rats were treated with paroxetine (10 mg/kg/d, s.c.) for 2 or 21 d. In both treatment groups, there was a marked prolongation of the effect of microiontophoretically applied 5-HT; however, in rats treated for 2 d, the prolongation was significantly greater than that observed in rats treated for 21 d. After the 21 d treatment with paroxetine and a 48 hr washout, the prolongation of the effect of microiontophoretically applied 5-HT by acute intravenous paroxetine was significantly reduced, suggesting a decrease in the number of 5-HT carriers. In keeping with this interpretation, following the same treatment regimen, there was a 50% and 60% reduction of the in vitro 3H-5-HT uptake in hippocampal and dorsal raphe slices, respectively, and a reduced effectiveness of paroxetine in blocking 3H-5-HT uptake in both regions. The determination of the binding parameters of 3H-paroxetine revealed that, in rats treated for 21 d with paroxetine (10 mg/kg/d, s.c.), following a 48 hr washout Kd values were unchanged but Bmax values were reduced by 70% and 60% in hippocampal and cortical membranes, respectively.