We have shown that Rab34/Rah participates in the promotion of macropinosome formation. Here we describe procedures for the analyses of intracellular localization and some functional properties of Rab34. Rab34 lacks a consensus sequence of the fourth motif for GTP/GDP binding and GTPase activities. Indeed, GTPase assay shows that wild-type Rab34 has extremely weak GTPase activity in vitro. However, Rab34 exhibits appreciable GTPase activity in vivo probably due to the presence of specific GTPase-activating protein (GAP) activity in cells. Specific intracellular localization of Rab34 is easily detected by the expression of epitope-tagged or enhanced green fluorescent protein (EGFP)-tagged protein. It is colocalized with actin filaments to membrane ruffles and membranes of nascent macropinosomes, which are formed from the ruffles. By contrast, Rab5 is not associated with the ruffles or nascent macropinosomes but present in endosomes at later stages. The function of Rab34 in macropinosome formation is analyzed by the transfection of wild-type, constitutively active, and dominant-negative mutants of Rab34 in fibroblasts followed by treatment with platelet-derived growth factor (PDGF) or phorbol ester. These analyses indicate that Rab34 is required for efficient macropinosome formation.