Annexin 2 is a Ca²⁺-regulated membrane protein and an F-actin-binding protein enriched at actin assembly sites both, on the plasma membrane and on endosomal vesicles. Here, we identify annexin 2 as a phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P₂)-interacting protein, thereby explaining this specific membrane association. Using the pleckstrin-homology (PH) domain of phospholipase Cδ1 fused to yellow fluorescent protein as a marker for PtdIns(4,5)P₂, we show that annexin 2 and its ligand p11 (S100A10) are targeted to sites of PtdIns(4,5)P₂ enrichment where F-actin accumulates. At the plasma membrane, adhesion of pedestal-forming enteropathogenic Escherichia coli induces a recruitment of 1-phosphatidylinositol-4-phosphate 5-kinase (PtdIns4P 5-kinase) and an enrichment of PtdIns(4,5)P₂ and annexin 2-p11 at sites of bacterial adhesion. Induction of PtdIns(4,5)P₂-enriched ruffles and PtdIns(4,5)P₂-positive, actin-coated vacuoles by Arf6-mediated activation of PtdIns4P 5-kinase also leads to a concomitant accumulation of the annexin 2-p11 complex and the PH domain. Binding studies with immobilized phosphoinositides and phosphoinositide-containing liposomes reveal that the purified annexin 2-p11 complex directly and specifically binds to PtdIns(4,5)P₂ with an affinity comparable to that of the PH domain of phospholipase Cδ1. Experiments using individual subunits identify annexin 2 as the PtdIns(4,5)P₂-binding entity. Thus, the direct interaction of annexin 2 with PtdIns(4,5)P₂ is a means of specifically recruiting the annexin 2-p11 complex to sites of membrane-associated actin assembly.