THE GTPase cycle is a versatile regulatory mechanism directing many cell functions(1), and Rab family members use it to regulate intracellular transport(2-4). Current models propose that GTP hydrolysis by Rah proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein(1-4). To measure the GTPase activity of Rab5 in endocytic membrane fusion(5), we engineered a mutant that preferentially binds xanthosine 5’-triphosphate (XTP), Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP (XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane Fusion and is reduced by its effector Rabaptin-5 (ref. 6). Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.