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Dopamine (DA) is a catecholamine neurotransmitter, which controls many physiological and psychological processes including reward, motivation, attention, and motor activity. Disruption of DA homeostasis has been implicated in a number of neurological diseases such as attention‐deficit hyperactivity disorder (ADHD), schizophrenia, depression, addiction, and Parkinson's disease. The dopamine transporter (DAT) is a plasma membrane protein that functions to move extracellular dopamine back into presynaptic neurons and is the primary means by which DA is cleared from the extraneuronal space. DAT is also a site of action for psychostimulant drugs such as cocaine and amphetamines, as well as therapeutic drugs such as methylphenidate and bupropion that are used to treat ADHD and depression respectively. Studying DAT function and its regulatory elements is crucial to better understand the pathophysiology of dopaminergic disorders, and to identify and improve treatments for these disorders. DAT activity is controlled by post translational modifications including ubiquitination, palmitoylation and phosphorylation. Phosphorylation occurs on two domains within the DAT N‐terminus: a protein kinase C (PKC) domain at the distal end containing serine 7 within a serine cluster and a proline‐directed mitogen activated protein kinase (MAPK) site at threonine 53 proximal to transmembrane domain 1. We have recently developed an antibody that specifically recognizes pT53 DAT and have utilized this tool to further investigate the functional importance of DAT T53 phosphorylation using fluorescent immunocytochemistry and confocal microscopy to determine subcellular localization of pT53 DAT in comparison with a pan antibody for total DAT (MAB16). Our preliminary results using two cell models (LLC‐PK 1 and N27 neurons) expressing rat DAT suggest that pT53 DAT is found at the plasma membrane and extensively throughout the cytoplasm displaying a distinct and intense punctate pattern while fluorescence detected with the pan MAB16 antibody was most intense at the plasma membrane with some intracellular punctate staining. These initial studies create a foundation for additional studies that will include fluorescent subcellular markers and treatments that modulate DAT phosphorylation at threonine 53 which may further our understanding of the functional relevance of DAT phosphorylation at this site and provide potential insight for involvement of dysregulation of DAT phosphorylation in dopaminergic disorders. Support or Funding Information Support: National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .