CD8 T-cells contribute to long-term protection against Listeria monocytogenes (Lm) infection by differentiating into memory T-cells. These rapidly respond to Ag or inflammation upon secondary infection. In this study we used CD8 T-cells from OT1 and CD4 T-cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing Lm (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as two days after infection. FACS analysis showed GZMB-Tom expression in un-divided CD8 T-cells, with its level increasing over 1-4 divisions. OT1 T-cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII positive cells. Twenty hours later, most OT1 T-cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom.Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T-cells responding either to Lm-OVA or Lm. Differences in the activation phenotype and in the splenic distribution of OT1 T-cells were observed, depending on the challenge. Notably, OTI T-cells with polarized granules were only observed after challenge with cognate Ag.This work showed that the GZMB-Tom KI-mice in which GZMB-Tom faithfully reproduced GZMB expression provide useful tools to dissect mechanisms leading to development of anti-bacterial effector and memory CD8 T-cells and reactivation of the memory response to cognate Ag or inflammatory signals.This article is protected by copyright. All rights reserved.