Rab proteins are small GTPases, localized to distinct cellular compartments, regulating specific steps of intracellular membrane trafficking. One member of the Rab family, Rab5, consists of three isoforms, Rab5a, Rab5b, and Rab5c, which have been shown to play an important role in early events of endocytosis. Using the yeast two-hybrid system, we have identified several cytosolic proteins that interact with the Rab5b Q79L (decreasing intrinsic and GTPase-activating protein-stimulated GTPase activities). Surprisingly, most positive clones were Rab5b or Rab5c, indicating that Rab5 could dimerize among extra-isoforms in the yeast two-hybrid system. In vitro and in vivo chemical cross-linking assays demonstrated that lipid-unmodified wild-type Rab5b purified from Escherichia coli, wild-type Rab5b, or a dominant active form Rab5b Q79L expressed in human 293T cells dimerized. Furthermore, the same assays using a Rab5b R81A substitution mutant showed that the Arg81 in the Switch II region [the second GTP/GDP binding motif (residues 74-93)] was essential for Rab5b dimerization. These results suggest that Rab5 isoforms can be dimerized depending upon the GTP-bound conformation, but independent upon a lipid modification.