The small GTPases of the Rho family play a key role in actin cytoskeletal organization. In plants, a novel Rho subfamily, called ROP (Rho of plants), has been found. In Arabidopsis, 12 ROP GTPases have been identified which differ mainly at their C-termini. To test the localization of two members of this subfamily (AtROP4 and AtROP6), we have generated translational fusions with the green fluorescent protein (GFP). Microscopic analysis of transiently transfected BY2 cells revealed a predominant localization of AtROP4 in the perinuclear region, while AtROP6 was localized almost exclusively to the plasma membrane. Swapping of the AtROP4 and AtROP6 C-termini produced a change in localization. As RhoGDIs are known to bind to the C-terminus of GTPases of the Rho family, we searched for Arabidopsis RhoGDI genes. We identified the AtRhoGDI1 gene and mapped it to chromosome 3. AtRhoGDI1 encodes a 22.5 kDa protein which contains highly conserved amino acids in the isoprene binding pocket and exhibits 29% to 37% similarity to known mammalian RhoGDI homologues. The AtRhoGDI1 gene was expressed in all tissues studied. Using the yeast two-hybrid system, we showed specific interaction of AtRhoGDI1 with both AtROP4 and AtROP6 as well as with their GTP-locked mutants, but not with a GTPase of the RAB family. Recombinant GST-AtRhoGDI1 could bind GFP-AtROP4 from transgenic tobacco BY2 cell extracts, confirming the interaction observed with the two-hybrid system.