Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this Study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific For Ras. (c) 2005 Elsevier Inc. All rights reserved.