Heterogeneous intracellular pathways and biochemical mechanisms are responsible for generating the glycoprotein complexes of peptide and major histocompatibility complex that are displayed on the surfaces of antigen-presenting cells for recognition by T lymphocytes. These pathways have a profound influence on the specificity of adaptive immunity and tolerance, as well as the context and consequences of antigen recognition by T cells in the thymus and periphery. The field of antigen processing and presentation has continued to advance since the publication of a focus issue on the topic in Nature Immunology in July 2004. Progress has been made on many fronts, including advances in understanding how proteases, accessory molecules and intracellular pathways influence peptide loading and antigen presentation in various cell types.