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.