Objective and hypothesis Monoamine transporters control extracellular monoamine levels in the brain by mediating their reuptake from the synaptic cleft, thereby terminating signal transduction in monoaminergic neurons. Previous studies of our lab have revealed that these transporters co-exist as monomers and oligomers at the plasma membrane. Transporter oligomerization has been suggested to play a role in transporter trafficking, drug-induced monoamine efflux, and drug tolerance development. However, many aspects regarding monoamine transporter oligomerization remain poorly understood. The objective of the present study was to investigate how psychostimulants affect monoamine transporter oligomerization for the first time on a single molecule level. Based on findings of other studies, the hypothesis was that substrate-type psychostimulants dissociate monoamine transporter oligomers whereas transporter inhibitors have no effect. Methods Transporter oligomerization was measured using single molecule microscopy in live transporter-transfected Chinese hamster ovary cells. The oligomeric state of fluorescently-labeled dopamine and serotonin transporter (DAT and SERT, respectively) was assessed in total internal reflection mode one hour after exposure to various transporter inhibitors and substrates. Results The substrates 3,4-methylenedioxymethamphetamine and mephedrone dissociated higher-order SERT oligomers, resulting in a statistically significant decrease in average oligomeric state (Student's t-test, P<0.05). Furthermore, both substrates reduced the kinetic trapping of SERT oligomers by phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast, none of the tested transporter inhibitors altered SERT oligomerization. Furthermore, none of the tested transporter substrates or inhibitors altered the oligomeric state of DAT. Conclusion The effect of psychostimulants on DAT and SERT oligomerization was for the first time investigated on a single molecule level in live cells. Substrate-type stimulants dissociated SERT higher-order oligomers whereas transporter inhibitors had no effect. None of the psychostimulants affected DAT oligomers. The effect of substrate-type psychostimulants therefore resembles that of direct phospholipase C agonists.