Progressive multifocal leukoencephalopathy (PML) is a severe infection of the central nervous system caused by the polyomavirus JC (JCV) that can occur in multiple sclerosis (MS) patients treated with natalizumab. Clinical management of patients with natalizumab-associated PML is challenging not the least because current imaging tools for the early detection, longitudinal monitoring and differential diagnosis of PML lesions are limited. Here we evaluate whether TSPO positron emission tomography (PET) imaging can be applied to monitor the inflammatory activity of PML lesions over time and differentiate them from MS lesions. For this monocenter pilot study we followed 8 patients with natalizumab-associated PML with PET imaging using the TSPO radioligand [18F]GE-180 combined with frequent 3 T MRI imaging. In addition we compared TSPO PET signals in PML lesions with the signal pattern of MS lesions from 17 independent MS patients. We evaluated the standardized uptake value ratio (SUVR) as well as the morphometry of the TSPO uptake for putative PML and MS lesions areas compared to a radiologically unaffected pseudo-reference region in the cerebrum. Furthermore TSPO expression in situ was immunohistochemically verified by determining the density and cellular identity of TSPO-expressing cells in brain sections from four patients with early natalizumab-associated PML as well as five patients with other forms of PML and six patients with inflammatory demyelinating CNS lesions (clinically isolated syndrome/MS). Histological analysis revealed a reticular accumulation of TSPO expressing phagocytes in PML lesions, while such phagocytes showed a more homogenous distribution in putative MS lesions. TSPO PET imaging showed an enhanced tracer uptake in natalizumab-associated PML lesions that was present from the early to the chronic stages (up to 52 months after PML diagnosis). While gadolinium enhancement on MRI rapidly declined to baseline levels, TSPO tracer uptake followed a slow one phase decay curve. A TSPO-based 3-dimensional diagnostic matrix taking into account the uptake levels as well as the shape and texture of the TSPO signal differentiated more than 96% of PML and MS lesions. Indeed, treatment with rituximab after natalizumab-associated PML in three patients did not affect tracer uptake in the assigned PML lesions but reverted tracer uptake to baseline in the assigned active MS lesions. Taken together our study suggests that TSPO PET imaging can reveal CNS inflammation in natalizumab-associated PML. TSPO PET may facilitate longitudinal monitoring of disease activity and help to distinguish recurrent MS activity from PML progression.