Endocytic proteins such as epsin, AP180, and Hip1R (SLa2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [Ptdlns(4,5)P-2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys(76) --> Ala(76), with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and Ptdlns(4,5)P-2 is essential for endocytosis mediated by clathrin-coated pits.