The translocator protein (TSPO) is expressed at low levels in the healthy human brain and is markedly upregulated in response to brain injury and inflammation. This increase in TSPO expression is correlated to the extent of microglial activation, making the measurement of TSPO density a useful indicator of active brain disease. Several classes of TSPO radioligands have therefore been developed and evaluated for use in PET, to track the progression and severity of neuroinflammatory disease. TSPO is also overexpressed in cancer and peripheral inflammation, making TSPO PET ligands possible candidates for the imaging of a multitude of pathologies. However, we currently possess a limited understanding about the molecular structure of TSPO and about the interaction of ligands with the protein. Furthermore, the incomplete characterization of multiple TSPO binding sites and the role of TSPO polymerization suggest that current interpretation of PET data may require further refinement.