The requirement of accessory cells for concanavalin A (Con A) activation of T cells suggests delivery of a separate costimulatory signal. However, the costimulatory pathways involved have not been identified. These studies assess the role of CD28–B7-mediated costimulation during T-cell activation by Con A. The B7-1/B7-2 binding protein CTLA4-Ig inhibited the proliferative response of primary lymph node cells to either Con A or soluble anti-CD3 mAb. This suppression was dose dependent and could be reversed by CD28 cross-linking. CTLA4-Ig also completely suppressed induction of interleukin-2 (IL-2) mRNA by Con A. CTLA4-Ig-mediated suppression was not due to blockade of the Con A ‘receptor(s)’ or of the primary activation signal (as measured by the intracellular calcium response). Although both B7-1 and B7-2 were up-regulated following Con A activation, each played a different role in proliferation and cytokine production. Individually, anti-B7-2 Fab partially inhibited the Con A response whereas anti-B7-1 Fab had no effect. However, the combination of anti-B7-1 and anti-B7-2 Fab completely suppressed proliferation and IL-2 production. Therefore, while a part of the Con A response requires B7-2, the remainder of the response can utilize either B7-1 or B7-2. Together, these results demonstrate that Con A activation of T cells requires the delivery of a separate costimulatory signal that is mediated almost entirely by the B7 receptors.