The present study evaluated high-speed chronoamperometry as a method for measuring the clearance of serotonin (5-HT) from extracellular space in vivo. Male Sprague-Dawley rats were anaesthetized and a Nafion-coated, carbon fiber electrode, attached to a multibarrel pipette, was lowered into the subgranular layer of the dentate gyrus, a region which receives dense serotonergic innervation, or the corpus callosum, a fiber tract relatively devoid of the 5-HT transporter (SERT). Serotonin, pressure ejected into these regions, produced replicable electrochemical signals. The amplitude and time course of the signals were significantly prolonged in the corpus callosum compared to the dentate gyrus. Similarly, signals produced by locally applied 5-HT in the dentate gyrus of rats following destruction of hippocampal serotonergic innervation with 5,7-dihydroxytryptamine (5,7-DHT), were significantly enhanced compared to those observed in control animals. The time course of the 5-HT signal was significantly prolonged by local application of the selective 5-HT reuptake inhibitor, fluvoxamine, into the dentate gyrus. By contrast, fluvoxamine did not modify the clearance of 5-HT when locally applied into the dentate gyrus of 5,7-DHT lesioned rats or into the corpus callosum of intact rats. Taken together, these data demonstrate that in intact rats, the SERT contributes to the clearance of exogenously applied 5-HT from the extracellular space. Under the experimental conditions used in this study, high-speed chronoamperometry proved to be a reliable method for directly measuring extracellular 5-HT and appears to be a valuable tool for the study of 5-HT clearance by the SERT in vivo.