Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which may operate in order to maintain appropriate levels of transmitter in the synaptic cleft. GABA and glycine transporters both interact with components of the neurotransmitter release, such as the SNARE protein syntaxin 1A, suggesting that protein-protein interactions are a common method for regulating members of the neurotransmitter transporter family, and thus, linking the release of transmitter to its subsequent re-uptake. In the present report, the interaction of syntaxin 1A with endogenous serotonin transporters (SERT) expressed in developing thalamocortical neurons is examined. Incubation of thalamocortical cultures with botulinum toxin C1, which specifically cleaves syntaxin 1A, decreased SERT function. Serotonin (5HT) saturation analysis showed that the effect of the toxin was to decrease maximum transport capacity with little change to the affinity of the transporter for 5HT. The 5HT uptake data were consistent with biotinylation experiments showing a decrease in the surface expression of SERT following toxin treatment. In addition, co-immunoprecipitation experiments showed that SERT and syntaxin 1A form a protein complex in these neurons. These data show that components of the transmitter release machinery interact with endogenously expressed amine transporters, and suggest a mechanism for the control of transmitter levels in disorders related to aminergic signaling.