Productive T cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. Naïve T cells are programmed to recirculate predominantly in secondary lymphoid tissue by non-specific cell:cell interactions and chemokines. In contrast, memory T lymphocytes must locate antigen location in non-lymphoid sites as their ability to respond is dependent on further antigen receptor triggering. The co-stimulatory molecule CD28 plays an essential role in the initiation of T cell-mediated immunity. We investigated the possibility that CD28 may also regulate migration of primed T cells to target tissue. In vitro, CD28-mediated signals enhanced T cell trans-endothelial migration, integrin clustering and integrin-mediated migration. In vivo, intact CD28 signalling – particularly PI3K activation – was required for efficient localization of primed T cell to non-lymphoid antigenic tissue. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T cell migration to extra-lymphoid tissue, which occurred independently of TCR-derived signals and homing-receptor expression. Finally, we observed that CD28- and CTLA-4-mediated signals exert opposite effects on T cell trafficking in vivo. These findings highlight a novel physiological function of the balance of co-stimulatory signals in the regulation of memory T cell trafficking, which has crucial implications for the therapeutic manipulation of these and other co-stimulatory molecules.