An electrogenic serotonin (5-HT) uptake process was characterized in the serotonergic Retzius-P cell synapse of the leech, and the simultaneous activation of this presynaptic reuptake and the postsynaptic response was monitored during evoked transmitter release. A presynaptic, Na(+)-dependent inward current upon application of 5-HT was isolated at membrane potentials between -80 and +60 mV. Its identification as a transmitter uptake current was confirmed by monitoring accumulation of the autofluorescent 5-HT analog 5,7-dihydroxytryptamine during activation of this current. To study the kinetics of 5-HT reuptake in functional synapses, transmitter release was stimulated by flash photolysis of the Ca(2+)-caging DM-nitrophen. The results demonstrate that reuptake activates with a minimal delay of less than a millisecond during synaptic transmission. It acts as a rapid transmitter removal system to determine the time course of the postsynaptic response and monitors the kinetics of transmitter clearance at the synaptic site.