During endocytosis, clathrin and the clathrin adaptor protein AP-2, assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane. One of these factors is Eps15, a clathrin-coat-associated protein that binds the alpha-adaptin subunit of AP-2. Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.