p60 217–225-specific CTLs are absent from mice immunized with the p60 218F mutant. BALB/c mice were immunized either with wild-type L. monocytogenes, the 92F mutant, or the p60 218F mutant. Six days later, spleen cells were stimulated in culture for 72 h, and the recovered cells were utilized as effector CTLs. 51CR-labeled RMAS-Kd cells were pulsed with a 10−9 M concentration of either the LLO 91–99 (solid bars) or p60 217–225 (stippled bars) peptide for 60 min and then added at 104 cells/well. Effector CTLs were added at an E/T ratio of 50:1. The assay was terminated 4 h later, and the percent 51Cr was determined. The data are representative of four experiments.