In 1985, the first members of the Rho GTPase family were identified. Over the next 10 years, rapid progress was made in understanding Rho GTPase signalling. Multiple Rho GTPases were discovered in a wide range of eukaryotes, and shown to regulate a diverse range of cellular processes, including cytoskeletal dynamics, NADPH oxidase activation, cell migration, cell polarity, membrane trafficking, and transcription. The Rho regulators, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs), were found through a combination of biochemistry, genetics, and detective work. Downstream targets for Rho GTPases were also rapidly identified, and linked to Rho-regulated cellular responses. In parallel, a wide range of bacterial proteins were found to modify Rho proteins or alter their activity in cells, many of which turned out to be useful tools to study Rho functions. More recent work has delineated where Rho GTPases act in cells, the molecular pathways linking some of them to specific cellular responses, and their functions in the development of multicellular organisms.