Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses. Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves. We find that two H2-K^d–restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics. Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates. p60 217-225 forms complexes with H2-K^d with a half-life >6 h, while p60 449-457 dissociates from H2-K^d with a half-life of ∼1 h. We find that p60 449-457–H2-K^d complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes. While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-K^d molecules maintain a remarkable capacity to bind new T cell epitopes. We find that intracellular H2-K^d molecules can bind new CTL epitopes for up to 3 h after their synthesis. Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells. We propose that the increased intracellular lability of peptide–MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.