This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The psychostimulant drug methamphetamine interferes with monoamine transporters to influence extracellular levels of monoamine neurotransmitters and alter brain monoaminergic tone. Trace amine-associated receptor 1 (TAAR1) is expressed in monoaminergic brain regions and is activated by methamphetamine in vitro. However, it is unclear whether TAAR1 is implicated in methamphetamine effects on monoamine transporters. This study used transfected HEK293 cells and brain synaptosomes to investigate the role of TAAR1 in methamphetamine modulation of the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT). We found that TAAR1 activation by methamphetamine significantly inhibited uptake and promoted efflux of (3H)dopamine, (3H)norepinephrine and (3H)serotonin by DAT, NET and SERT, respectively, in transfected cells. Similarly, methamphetamine significantly inhibited uptake and induced efflux of (3H)dopamine and (3H)serotonin in striatal and (3H)norepinephrine in thalamic synaptosomes of monkeys and wild type mice, but it had no such effect in TAAR1 knockout mouse synaptosomes. Both the PKA inhibitor H89 and PKC inhibitor Ro32-0432 blocked uptake inhibition caused by methamphetamine, and Ro32-0432 blocked methamphetamine-induced efflux in wild type mouse synaptosomes. TAAR1 signaling in response to methamphetamine was unaffected by monoamine autoreceptors (D2s, alpha2A, alpha2B, 5HT1A and 5HT1B) co-expressed in transfected cells, and binding assays showed that methamphetamine had extremely low binding affinity for the monoamine autoreceptors in contrast to dopamine, norepinephrine and serotonin. Together, these results provide the first evidence that methamphetamine interacts with TAAR1 but not monoamine autoreceptors to alter uptake and efflux functions of brain monoamine transporters via phosphorylation-dependent pathways.