Endocytosis is regulated by several GTPases including Rab5 and one or more heterotrimeric G proteins. We show here that Rab5, in the GTPγS (guanosine 5'-O-(thiotriphosphate)-bound form, fully supports in vitro endosome fusion, indicating that GTP hydrolysis is not required, whereas Rab5:S34N and Rab5:N133I, mutants unable to bind GTP, are potent inhibitors of endosome fusion. Double mutants (Rab5:S34N/ΔC4 and Rab5:N133I/ΔC4) lacking the C- terminal prenylation site were inactive, indicating that prenylation is required. Endosomes became resistant to the inhibitory effects of Rab5:S34N by preincubating the vesicles with cytosol prior to the addition of the inhibitor. The acquisition of resistance to Rab5:S34N was more rapid than to N-ethylmaleimide, indicating that Rab5 mutants are early acting. G(βγ) subunits of heterotrimeric G proteins block endosome fusion. However the effect of G(βγ) was abrogated by Rab5·GTPγS, indicating that a heterotrimeric G protein may operate upstream of Rab5.