Productive T-cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. The costimulatory 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 transendothelial migration, integrin clustering, and integrin-mediated migration. In vivo, T cells bearing a mutation in the CD28 cytoplasmic domain, which abrogates PI3K activation, displayed normal clonal expansion but defective localization to antigenic sites following antigenic rechallenge. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T-cell migration to extra-lymphoid tissue, which occurred independently of T-cell receptor (TCR)–derived signals and homing-receptor expression. Finally, we provide evidence that CD28- and CTLA-4–mediated signals exert opposite effects on T-cell trafficking in vivo. These findings highlight a novel physiologic function of CD28 that has crucial implications for the therapeutic manipulation of this and other costimulatory molecules.