MHC class I molecules function to present peptides comprised of eight to 11 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory to understand how cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and non-proteasomal proteases to peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.