Perturbation of several distinct T cell molecules, including the CD3/TCR complex, CD7, and CD28, activates phosphatidylinositol 3-kinase (PI3-K), but a clear consensus on the role of PI3-K in T cell activation has yet to emerge. We report here that CD3 mAb-induced IL-2 production by CD4+ T cells from DO11.10 TCR-alphabeta-transgenic mice is refractory to the potent PI3-K inhibitor, wortmannin, demonstrating that activation under these conditions is independent of PI3-K. In marked contrast, wortmannin substantially inhibits IL-2 production elicited by Ag (OVA(323-339) peptide) presented by appropriate APCs (syngeneic B7+ B cell blasts) and blocks Ag-induced differentiation of naive CD4+ DO11.10 T cells into IL-4-producing cells. Wortmannin inhibits Ag-induced conjugate formation between T cells and B7+ B cell blasts. Because T cell activation by Ag requires stable interactions with APCs, this inhibitory effect on conjugate formation may underlie the ability of wortmannin to block Ag-induced IL-2 production and differentiation.