Although under certain conditions an association with phosphatidylinositol 3'-kinase (PI3-K) appears to be critical for CD28 signaling, mutation of the PI3-K binding site (Tyr 170) does not alter the costimulatory ability of murine CD28 (mCD28) in Jurkat T cells. To define the structural requirements for this PI3-K-independent signaling, we expressed a series of mCD28 mutants in Jurkat. Mutation to Phe of all four cytoplasmic Tyr residues together (ALL F mutant) greatly reduced the ability of mCD28 to augment IL-2 production. Isolated re-constitution of Tyr 188, but not 170, 185, or 197, restored the ability of ALL F mCD28 to deliver a costimulus. Thus, a signal based upon Tyr 188 can deliver a costimulus for the enhancement of IL-2 production by Jurkat cells.