The high affinity serotonin transporter (SERT) constitutes the principal pathway for removal of serotonin (5-HT) from extracellular fluid of brain, but evidence indicates that other transporters may also be involved in this process. We recently reported the cloning of a novel plasma membrane monoamine transporter (PMAT), which is abundantly expressed in the human brain and avidly transports 5-HT [Engel K, Zhou M, Wang J. Identification and characterization of a novel monoamine transporter in the human brain. J Biol Chem 2004;279:50042-9]. In this study, we evaluated whether PMAT contributes to total human brain uptake of 5-HT using a hybrid depletion approach in Xenopus laevis oocytes. We also examined whether PMAT interacts with selective serotonin reuptake inhibitors (SSRIs) using MDCK cells stably expressing recombinant human PMAT. Microinjection of total human brain poly(A)(+) mRNA into oocytes elicited approximately 2.5-3-fold increase in 5-HT uptake. Pre-hybridization of poly(A)(+) mRNA with PMAT or SERT antisense oligonucleotides significantly reduced mRNA-induced 5-HT uptake. An additive inhibitory effect was observed when poly(A)(+) mRNA was co-hybridized with both PMAT and SERT antisense oligonucleotides. In contrast, mRNA-induced 5-HT uptake was not affected by pre-hybridization with sense oligonucleotides. These data suggest that functional transcripts of PMAT are present in the human brain, and the PMAT transporter may be significantly involved in brain uptake of 5-HT. All five tested SSRIs inhibited PMAT with IC(50) values ranging from 11 to 116 microM, which are much greater than clinically encountered concentrations, suggesting that PMAT activity is minimally affected by SSRI therapies.