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In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and α-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1–3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity [4]. The membrane association of early endosomal antigen 1 (EEA1), a specific marker of early endosomes [5,6], has recently been shown to be similarly dependent on PI 3-kinase activity [7], and we therefore postulated that it might be involved in endosome fusion. Here, we present evidence that EEA1 has an important role in determining the efficiency of endosome fusion in vitro. Both the carboxy-terminal domain of EEA1 (residues 1098–1411) and specific antibodies against EEA1 inhibited endosome fusion when included in an in vitro …