Homology searches toward the EST databases of Dictyostelium discoideum identified two putative Rho GDP-dissociation inhibitors (RhoGDIs), RhoGDI1 and RhoGDI2. In this study, the roles of RhoGDI1 in cytokinesis were examined. The RhoGDI1-null Dictyrostelium strains produced by homologous recombination were viable but generated multinucleate giant cells in suspension culture, suggesting that RhoGDI1 is involved in cytokinesis, The expression of green fluorescent protein (GFP)-tagged RhoGDI1 complemented the defects of the RhoGDI1-null cells, and the GFP-RhoGD1 I is predominantly present in cytoplasm of the cell-like yeast RhoGDI. Of 15 Rho family GTPases in Dictyostelium currently known, Dietyostelium versions of Rac1 proteins (Rac1A, Rac1B, and Rac1Q and RacE that are reportedly involved in Dictyostelium cytokinesis, showed two-hybrid interactions with RhoGDI1 as well as human and yeast Cdc42. These results suggest that RhoGDI1 is involved in cytokinesis of Dictyostelium through the regulation of Rho family GTPases Rac1s and/or RacE. (C) 2002 Elsevier Science (USA). All rights reserved.