Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.