words)Major histocompatibility complex class II (MHCII) 1 molecules display peptides on antigen-presenting cells (APCs) for inspection by CD4+ T cells. MHCII surface levels and life span are regulated post-translationally by peptide loading and ubiquitindependent lysosomal targeting, but proteases responsible for MHCII protein degradation remain unidentified. Here, we examined the role of aspartyl proteases in MHCII protein degradation and characterised the form of MHCII that is degraded. Exposure of immature monocyte-derived dendritic cells (MoDCs) and KG-1 acute myeloid leukaemia cells to the aspartyl protease inhibitor, pepstatin A (PepA), caused accumulation of human leukocyte antigen (HLA)-DR molecules in intracellular vesicles. Statistically significant PepA effects on MHCII protein expression were also observed in murine APCs. In KG-1 cells, cathepsin D (CatD) was the sole expressed aspartyl protease, and shRNA-mediated knockdown ablated the PepA effect, providing formal proof of CatD involvement. In vitro, CatD initiated specific cleavage of recombinant DR at F54, a site flanking the peptide-binding groove. Immunochemical characteristics of PepA-rescued DR molecules in KG-1 cells were consistent with selective CatD attack on HLA-DR molecules that lack association with the MHCII chaperone, invariant chain, or with stably bound peptide. We propose that CatD has a critical role in the selective lysosomal disposal of mature HLA-DR molecules that have lost, or never acquired, bound peptide, explaining how MHCII protein life span is coupled to peptide loading.