CD8⁺ T cells play a central role in the resolution and containment of viral infections. A key effector function of CD8⁺ T cells is their cytolytic activity toward infected cells. Here, we studied the regulation of cytolytic activity in naive, effector, and central versus effector memory CD8⁺ T cells specific for the same glycoprotein-derived epitope of lymphocytic choriomeningitis virus. Our results show that the kinetics of degranulation, assessed by a novel flow cytometric based assay, were identical in effector and both subsets of memory CD8⁺ T cells, but absent in naive CD8⁺ T cells. However, immediate cytolytic activity was most pronounced in effector T cells, low in effector memory T cells, and absent in central memory T cells, correlating with the respective levels of cytolytic effector molecules present in lytic granules. These results indicate that an inherent program of degranulation is a feature of antigen-experienced cells as opposed to naive CD8⁺ T cells and that the ability of CD8⁺ T cells to induce target cell apoptosis/death is dependent on granule protein content rather than on the act of degranulation itself. Furthermore, these results provide a potential mechanism by which central memory CD8⁺ T cell–mediated death of antigen-presenting cells within the lymph node is avoided.