Cell adhesion and migration are regulated through the concerted action of cytoskeletal dynamics and adhesion proteins, the activity of which is governed by RhoGTPases. Specific RhoGTPase signaling requires spatio-temporal activation and coordination of subsequent protein-protein and protein-lipid interactions. The nature, location and duration of these interactions are dependent on polarized extracellular triggers, such as cell-cell contact, and intracellular modifying events, such as phosphorylation. RhoA, RhoB, and RhoC are highly homologous GTPases that, however, succeed in generating specific intracellular responses. Here, we discuss the key features that contribute to this specificity. These not only include the well-studied switch regions, the conformation of which is nucleotide- dependent, but also additional regions and seemingly small differences in primary sequence that also contribute to specific interactions. These differences translate into differential surface charge distribution, local exposure of amino acid side-chains and isoform-specific post-translational modifications. The available evidence supports the notion that multiple regions in RhoA/B/C cooperate to provide specificity in binding to regulators and effectors. These specific interactions are highly regulated in time and space. We therefore subsequently discuss current approaches means to visualize and analyze localized GTPase activation using biosensors that allow imaging of isoform-specific, localized regulation.