Rho GTPases regulate cytoskeletal and cell adhesion dynamics and thereby coordinate a wide range of cellular processes, including cell migration, cell polarity and cell cycle progression. Most Rho GTPases cycle between a GTP-bound active conformation and a GDP-bound inactive conformation to regulate their ability to activate effector proteins and to elicit cellular responses. However, it has become apparent that Rho GTPases are regulated by post-translational modifications and the formation of specific protein complexes, in addition to GTP–GDP cycling. The canonical regulators of Rho GTPases—guanine nucleotide exchange factors, GTPase-activating proteins and guanine nucleotide dissociation inhibitors—are regulated similarly, creating a complex network of interactions to determine the precise spatiotemporal activation of Rho GTPases.