Rho GTPases are ubiquitously expressed across the eukaryotes where they act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. Activation enables Rho GTPases to interact with a multitude of effectors that relay upstream signals to cytoskeletal and other components, eliciting rearrangements of the actin cytoskeleton and diverse other cellular responses. In Dictyostelium the Rho family comprises 15 members. Some of them (Rac1a/b/c, RacF1/F2, RacB) are members of the Rac subfamily, and one, RacA, belongs to the RhoBTB subfamily, however the Rho and Cdc42 subfamilies are not represented. Dictyostelium Rho GTPases regulate actin polymerization, cell morphology, endocytosis, cytokinesis, cell polarity and chemotaxis. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) modulate the activation/inactivation cycle of the GTPases. In addition, guanine nucleotide-dissociation inhibitors (GDIs) regulate cycling of the GTPases between membranes and cytosol. Members of these three classes of regulatory molecules along with some effectors have been identified in Dictyostelium during the last years and their role in Rho signaling pathways has been investigated.