Murine infection with the intracellular bacterium Listeria monocytogenes elicits MHC class I-restricted CTL with specificity for multiple bacterial peptides. The variety and relative abundance of self-peptides bound by MHC molecules make identification of pathogen-derived peptides difficult. In this report, the sequence of a pathogen-derived CTL epitope is determined by direct analysis of peptides extracted from MHC class I molecules. The epitope, p60 217-225, is presented to L. monocytogenes-specific CTL by the H-2Kd MHC class I molecule and is derived from p60, a secreted invasion-associated protein. Quantitation of p60 217-225 in infected cells shows that this epitope is detectable within 2 h of infection and, after a 9-h infection, there are over 3000 epitopes per infected cell. This contrasts with listeriolysin 91-99, the other major L. monocytogenes epitope, which is present in quantities below 200 epitopes per cell until 5 h of infection and reaches 800 epitopes per cell 9 h after infection. This report shows that identifying new T lymphocyte epitopes by direct sequence analysis of peptides isolated from MHC molecules is feasible. Furthermore, kinetic and quantitative analysis of T cell epitopes in infected cells is a useful approach to investigate the multispecific CTL response to complex intracellular pathogens.