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The adaptive immune system, particularly T cells and more recently B cells, is considered to play a major role in the pathogenesis of multiple sclerosis (MS). In addition to adaptive immune mechanisms, innate central nervous system (CNS) immunity, particularly mediated by microglia, may play a key role in MS pathogenesis. Microglial activation has been demonstrated throughout the MS disease course and at various locations in the CNS, including white and gray matter lesions as well as normal appearing white and gray matter. Activated microglia overexpress an 18 kilodalton mitochondrial translocator protein (TSPO), which is otherwise expressed at low levels during the resting state. Several positron emission tomography (PET) ligands targeting TSPO have been developed to enable the assessment of microglial activation. In this article, we review the biological basis, methodological aspects, and current status of TSPO-PET imaging in MS and discuss future research directions.