The granular exocytosis pathway is one mechanism by which NK cells and CTLs induce cytolysis of target cells. Triggering through adhesion molecules such as CD2 and LFA-1 as well as Fc gammaRIII (CD16) can invoke this pathway. CD2 is a cell surface glycoprotein present on CTLs and NK cells that plays an important role in both cellular adhesion and signal transduction. Here we report that cross-linking of CD2 as well as CD16 by immobilized Abs enhances granular exocytosis in an NK cell line, NK3.3. Herbimycin, a protein tyrosine kinase (PTK) inhibitor, or wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-K), inhibited completely or almost completely CD2- or CD16-mediated granular exocytosis, suggesting the involvement of protein tyrosine kinases and PI 3-K in both CD2- and CD16-mediated granular exocytosis. We also observed that cross-linking of CD2 as well as CD16 enhances p72syk tyrosine kinase activity, and this enhancement correlated well with the increased tyrosine phosphorylation of several cellular proteins, including the adapter protein Shc. Furthermore, we have observed that cross-linking of CD2 as well as CD16 enhances the PI 3-K activity associated with the tyrosine-phosphorylated cellular proteins and Shc. These results provide insight into the signaling pathways by which triggering of CD2 and CD16 on NK cells leads to cytolysis of target cells.