In CD8+ T cells, engagement of the TCR with agonist peptide:MHC molecules causes dynamic redistribution of surface molecules including the CD8 coreceptor to the immunological synapse. CD8 associates with the Src-family kinase (SFK) Lck, which, in turn, initiates the rapid tyrosine phosphorylation events that drive cellular activation. Compared with naive T cells, Ag-experienced CD8+ T cells make shorter contacts with APC, are less dependent on costimulation, and are triggered by lower concentrations of Ag, yet the molecular basis of this more efficient response of memory T cells is not fully understood. In this article, we show differences between naive and Ag-experienced CD8+ T cells in colocalization of the SFKs and their negative regulator, C-terminal Src kinase (Csk). In naive CD8+ T cells, there was pronounced colocalization of SFKs and Csk at the site of TCR triggering, whereas in Ag-experienced cells, Csk displayed a bipolar distribution with a proportion of the molecules sequestered within a cytosolic area in the distal pole of the cell. The data show that there is differential redistribution of a key negative regulator away from the site of TCR engagement in Ag-experienced CD8+ T cells, which might be associated with the more efficient responses of these cells on re-exposure to Ag.