The small GTPases of the Rho family, which number more than 20 in humans, carry out diverse signalling functions. The Rho GTPase family is thought to have expanded through a series of gene duplications, but how do new family members evolve to perform distinct tasks? On page 1065, Hans-Peter Schmitz and colleagues address this question by studying two closely related Rho proteins in the fungus Ashbya gossypii. The authors show that Rho1a and Rho1b play distinct cellular roles (actin regulation and cell-wall biosynthesis, respectively) and localise to different sites within the cell. They go on to demonstrate that a single amino acid polymorphism is largely responsible for these differences. When the key histidine residue of Rho1a is replaced by tyrosine (which is found at this position in Rho1b), the mutant protein localises to the cell periphery, as Rho1b does, and rescues lethality in a strain of A. gossypii that lacks Rho1b. Crucially, the polymorphism is also responsible for the differential affinities of the proteins for Sac7 and Lrg1, two GTPase-activating proteins (GAPs) that regulate Rho GTPase activity. The authors suggest, therefore, that the divergent functions of Rho1a and Rho1b result from their differential regulation by GAPs.