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HLA-DO, a non-classical class II major histocompatibility complex (MHCII), competitively inhibits HLA-DM in B cells. B cells lower DO levels during antigen-mediated activation in germinal centers, implying a regulatory function of DO in resting B cells. We transfected T2 (T/B hybrid) DR4.DM cells with DO and used index FACS to measure single cell variation in DO:DM ratios. High DO:DM ratios allowed groove occupancy by class II-associated Ii peptides (CLIP), and low DO:DM ratios enabled CLIP removal. 3D structured illumination microscopy revealed DMDO concentrated in LAMP1+ endosomes. Both DM+DO+ and DM−DO− cells express CLIP/DR complexes, but a larger fraction of CLIP/DR localizes to LAMP1+ endosomes in DM+DO+ cells. Using memory and naïve human tonsillar B cells, we saw significant downregulation of DO after cell activation. CLIP/MHCII density relative to free DM (estimated by 1-DO/DM) was generally higher in resting vs. activated B cells, especially in memory cells. This memory B cell phenotype primarily reflected higher intracellular CLIP/MHCII; upon BCR-mediated stimulation, total CLIP/MHCII fell to levels in activated naïve cells. We hypothesized that higher CLIP/MHCII and DO in resting memory B cells sets a threshold for the activation needed to efficiently recruit Tfh and selects for cells with higher affinity BCR. Using antigen stimulation of 2 clonal B cell lines expressing BCRs with different affinities for GAD65, we confirmed that, in this system, BCR affinity correlates with the amount of reduction in total CLIP/MHCII after activation. A positive correlation between BCR affinity and the extent of peptide exchange on MHCII is likely mediated by the effects of BCR affinity on signaling and antigen delivery.