¹¹C-PBR28 PET can detect the 18-kDa translocator protein (TSPO) expressed within macrophages. However, quantitative evaluation of the signal in brain tissue from donors with multiple sclerosis (MS) shows that PBR28 binds the TSPO with high affinity (binding affinity [K_i], ∼4 nM), low affinity (K_i, ∼200 nM), or mixed affinity (2 sites with K_i, ∼4 nM and ∼300 nM). Our study tested whether similar binding behavior could be detected in brain tissue from donors with no history of neurologic disease, with TSPO-binding PET ligands other than ¹¹C-PBR28, for TSPO present in peripheral blood, and with human brain PET data acquired in vivo with ¹¹C-PBR28. The affinity of TSPO ligands was measured in the human brain postmortem from donors with a history of MS (n = 13), donors without any history of neurologic disease (n = 20), and in platelets from healthy volunteers (n = 13). Binding potential estimates from thirty-five ¹¹C-PBR28 PET scans from an independent sample of healthy volunteers were analyzed using a gaussian mixture model. Three binding affinity patterns were found in brains from subjects without neurologic disease in similar proportions to those reported previously from studies of MS brains. TSPO ligands showed substantial differences in affinity between subjects classified as high-affinity binders (HABs) and low-affinity binders (LABs). Differences in affinity between HABs and LABs are approximately 50-fold with PBR28, approximately 17-fold with PBR06, and approximately 4-fold with DAA1106, DPA713, and PBR111. Where differences in affinity between HABs and LABs were low (∼4-fold), distinct affinities were not resolvable in binding curves for mixed-affinity binders (MABs), which appeared to express 1 class of sites with an affinity approximately equal to the mean of those for HABs and LABs. Mixed-affinity binding was detected in platelets from an independent sample (HAB, 69%; MAB, 31%), although LABs were not detected. Analysis of ¹¹C-PBR28 PET data was not inconsistent with the existence of distinct subpopulations of HABs, MABs, and LABs. With the exception of ¹¹C-PK11195, all TSPO PET ligands in current clinical application recognize HABs, LABs, and MABs in brain tissue in vitro. Knowledge of subjects’ binding patterns will be required to accurately quantify TSPO expression in vivo using PET.