The highly conserved GTP-binding proteins Cdc42 and Rac1 regulate cytokinesis, establishment of cell polarity and vesicular trafficking. In the dimorphic fungus Ustilago maydis, Rac1 is required for cell polarity and budding, while Cdc42 is essential for cell separation during cytokinesis. The same cell separation defect is also observed in mutants that lack Don1, a guanine nucleotide exchange factor (GEF) of the Dbl family. We have generated a series of chimeric GTP-binding proteins consisting of different portions of Cdc42 and Rac1. In vivo complementation analysis revealed that a short region encompassing amino acids 41-56 determines signalling specificity. Remarkably, substitution of a single amino acid at position 56 within this specificity domain is sufficient to confer Cdc42 function to Rac1 in vivo. Expression of Rac1(W56F) in Delta cdc42 mutant cells resulted in complementation of the cell separation defect. In vitro GDP/GTP exchange assays demonstrated that the Dbl family GEF Don1 is highly specific for Cdc42 and cannot activate Rac1. However, if Rac1(W56F) is used as a substrate, Don1 is able to stimulate GDP/GTP exchange. Together these data indicate that activation by the GEF Don1 is an important determinant of Cdc42-specific signalling in vivo.