Major histocompatibility complex (MHC) class II-associated antigen presentation involves an array of interacting molecules. CD74, the cell surface isoform of the MHC class II-associated invariant chain, is one such molecule; its role remains poorly defined. To address this, we have employed a high-resolution single-particle imaging method for quantifying the colocalization of CD74 with human leukocyte antigen (HLA)-DR molecules on human fibroblast cells known for their capacity to function as antigen-presenting cells. We have also examined whether the colocalization induces internalization of HLA-DR using HA(307-319), a "universal" peptide that binds specifically to the peptide-binding groove of all HLA-DR molecules, irrespective of their alleles. We have determined that 25 ± 1.3% of CD74 and 17 ± 0.3% of HLA-DR are colocalized, and the association of CD74 with HLA-DR and the internalization of HLA-DR are both inhibited by HA(307-319). A similar inhibition of HLA-DR internalization was observed in freshly isolated monocyte-derived dendritic cells. A key role of CD74 is to translocate HLA-DR molecules to early endosomes for reloading with peptides prior to recycling to the cell surface. We conclude that CD74 regulates the balance of peptide-occupied and peptide-free forms of MHC class II at the cell surface.