Abstract Despite effective anti-retroviral therapy (ART) that maintains HIV at non-detectable levels in plasma, HIV is not eradicated. When individuals are off ART, or during viral blips, CNS viral reservoirs can quickly rebound. We and others have found that a population of CNS perivascular macrophages (PVMs) function as a major target for HIV and SIV infection in the CNS, and the viral reservoir that persists with ART. Intracisternal (i.c.) injection of superparamagnetic iron oxide nanoparticles (SPIONs) demonstrate PVMs take up SPIONs within the CNS, accumulate with SIV infection, and traffic out of the CNS where they are found in cervical draining lymph nodes (cLNs), dorsal root ganglia, spleen, and bone marrow. Importantly, SPION-labeled CD163+ macrophages in the cLN can be productively infected with SIV as evidenced by SIV-p27 immunoreactivity. It is our overall hypothesis that an identifiable population of PVMs in the CNS functions as a cellular reservoir of rebound HIV and SIV during ART, and after ART cessation, and these cells can leave the CNS with virus that potentially reseeds the periphery. To test our hypothesis, we propose to use a CNS-penetrant colony-stimulating factor 1 receptor (CSF1R) inhibitor (BLZ945) that ablates these reservoir-reseeding CNS PVM early (3 months after ART initiation) and late (5 months after ART initiation) during ART in virally suppressed macaques, and in animals undergoing ART cessation. We propose to use 2 different fluorescently tagged SPIONs, injected intra-CSF just prior to early and late BLZ945 treatments, in order to define the role of resident and repopulated PVMs to function as a viral reservoir of SIV, and block their ability emigrate with virus. We propose to test our hypothesis with two Specific Aims: Aim 1 will determine the extent to which CSF1R blockade can eradicate SIV in the brain and block lymphatic-dependent reseeding of virus from the CNS to the periphery in the presence of ART; and Aim 2 will determine whether CSF1R blockade can prevent reactivation of SIV reservoirs in the brain and repopulation of viral reservoirs from the CNS to the periphery after ART interruption.