Human alpha-synuclein (alpha-Syn) is instrumental in maintaining homeostasis of monoamine neurotransmitters in brain, through its trafficking, and regulation of the cell surface expression and, thereby, activity of dopamine, serotonin and norepinephrine transporters. Here we have investigated whether other members of the synuclein family of proteins, gamma-synuclein (gamma-Syn) and beta-synuclein (beta-Syn) can similarly modulate the serotonin transporter (SERT). In Ltk(-) cells co-transfected with SERT and gamma-Syn, gamma-Syn reduced [(3)H]5-HT uptake, in a manner dependent on its expression levels. The decrease in SERT activity was via decreased V(max) of the transporter, without change in K(m), compared to cells expressing only SERT. By contrast, beta-Syn co-expression failed to alter SERT uptake activity, and neither the V(max) nor the K(m) was changed in the presence of beta-Syn. gamma-Syn modulation of SERT was only partial, with a maximal approximately 27% decrease in SERT activity seen even at high expression levels of gamma-Syn. By contrast, alpha-Syn attenuated SERT activity by approximately 65% at identical expression levels as gamma-Syn. Co-immunoprecipitation studies showed the presence of heteromeric protein:protein complexes between gamma-Syn or alpha-Syn and SERT, while beta-Syn failed to physically interact with SERT. Both alpha-Syn and gamma-Syn colocalized with SERT in rat primary raphae nuclei neurons. These studies document a novel physiological role for gamma-Syn in regulating 5-HT synaptic availability and homeostasis, and may be of relevance in depression and mood disorders, where SERT function is dysregulated.