In this report, the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb. We demonstrate that while both anti-CD3 and anti-CD28 antibodies induced activation of phospholnositide (PI) 3-kinase, the kinetics of activation differed. Anti-CD28 produced a sustained activation of PI 3-kinase while anti-CD3 induced activation was transient. Both B7-1 and B7-2 could induce prolonged activation of PI 3-kinase. The co-stimulatory effects of B7-1 and B7-2 were dependent on CD28 cross-linking, based on complete inhibition of PI 3-kinase activation by CD28 antibody Fab fragments. While Jurkat T cells co-stimulated with anti-CD3 and B7-1 or B7-2 secreted high levels of IL-2, there were distinct effects of anti-CD28 mAb and B7-1 or B7-2 on IL-2 secretion in conjunction with protein kinase C activation. To assess functional effects of CD28 ligation, pharmacologic inhibitors of PI 3-kinase were evaluated. In Jurkat cells, efficient inhibition of PI 3-kinase activation after B7-2 stimulation was achieved using wortmannin; however, we observed a surprising increase in IL-2 secretion after B7 or anti-CD28 stimulation. The effect of wortmannin was concentration dependent. Moreover, the effect was specific for receptor-mediated activation as wortmannin did not enhance phorbol ester plus lonomycin-induced IL-2 secretion. Another inhibitor of PI 3-kinase, LY294002, also resulted in augmentation of anti-CD28-induced IL-2 secretion by Jurkat cells. The effects of wortmannin on IL-2 secretion were also examined in primary T cells. In marked contrast, wortmannin resulted in a potent inhibition of anti-CD3 plus B7-1 or anti-CD28-induced IL-2 secretion while phorbol ester plus lonomycin-induced IL-2 secretion was wortmannin resistant. Together these observations demonstrate that signal transduction by both B7-1 and B7-2 involves PI 3-kinase, and that PI 3-kinase or other wortmannin-sensitive targets are important for IL-2 secretion. Finally, treatment of Jurkat cells with PI 3-kinase inhibitors alone was sufficient to induce low levels of IL-2 secretion. This is consistent with the notion that a wortmannin-sensitive target such as PI 3-kinase may down-regulate IL-2 secretion in Jurkat cells.