AbstractCD28 is a 44‐kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter‐receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3‐kinase (PI3‐K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28‐transfected cells or pre‐activated T cells. In addition, recent reports propose that CD28‐B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28‐B7.1 and CD28‐B7.2 interactions induce the association of PI3‐K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine‐phosphorylated CD28 and does not require pre‐activation by CD3‐T cell receptor. Worthmannin, a specific inhibitor of PI3‐K enzymatic activity within the nanomolar range also inhibits the interleukin‐2 production induced by costimulation mediated by either the B7.1‐ and B7.2‐transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC50of wortmannin being 25 and 110 nM, respectively, which could suggest differences in the