The interaction between CD28 on T cells with CD80 (B7-1) and CD86 (B7-2) on APCs is considered to be of critical importance for primary T cell activation both in vivo and in vitro. The relative importance of this co-stimulatory signal in memory T cell activation is, however, less clear, and was therefore studied by in vitro experiments on T cell responses to soluble recall antigens using peripheral blood mononuclear cells or T cell clones. Our data demonstrate that B7-2 represents the major co-stimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens, as evidenced by the effects of anti-B7-1 and anti-B7-2 on T cell proliferation as well as on IL-2 and INF-y production. Since CTLA-4-lg and anti-CD28 Fab fragments had similar inhibitory effects to the combination of anti-B7-1 plus anti-B7-2, the involvement of a third co-stimulatory CD28/CTLA-4 ligand is unlikely. Despite the strong effects of B7-blocking agents, a variable fraction of the memory T cells was resistant to inhibition. Moreover, T cell clones or in vitro preactivated T cells could efficiently be restimulated by soluble antigens on autologous APCs in the absence of B7-1 or B7-2 co-stimulation. These data show that most memory T cells that are freshly isolated from the blood are still dependent on CD28 triggering for their activation. However, recently activated T cells can apparently bypass the requirement for B7 and use other co-stimulatory signals for reactivation, a finding with important implications for the development of immunosuppressive strategies.