Exosomes secreted by dendritic cells (DCs) contain MHC-I, MHC-II, and other accessory molecules required for antigen presentation to T cells. Previous studies have shown that exosome MHC-I "indirectly" loaded by adding peptides to DC cultures are immunogenic. However, analysis of peptide binding was not performed to link T-cell-stimulating activity with the amount of MHC-I/peptide complexes on the exosomes. In this study, we measured peptide binding to MHC-I under different loading conditions and tested the exosomes' potencies in T-cell activation assays. We demonstrate that MHC-I on purified exosomes can be directly loaded with peptide at much greater levels than indirect loading. The direct loading method performed in mildly acidic conditions was effective even in the absence of exogenous beta2m. This increase in peptide binding greatly enhanced exosome potency, allowing us to further study the biologic activity of exosomes in vitro. In the presence of antigen-presenting cells (APC), exosomes directly loaded with the HLA-A2 restricted MART1 tumor peptide stimulated an HLA-A2/MART1 specific T-cell line. The T cells responded to exosomes using HLA-A2neg APC, demonstrating transfer of functional MHC-I/peptide complexes and not peptide alone to APC. MHC-II molecules, which are abundantly expressed on DC exosomes, were also functionally loaded under the same conditions as MHC-I. This feature allows for delivery of multiple peptide antigens that can stimulate both CD8+ cytotoxic T cells as well CD4+ T helper cells critical for an effective antitumor response. The optimized loading conditions and the ability to transfer both MHC-I and MHC-II antigens to APC have led to the development of exosomes as an "acellular" immunotherapy approach currently being tested in clinical trials.