In the dimorphic fungus Ustilago maydis the Rho-family GTP-binding protein Cdc42 and the Ste20-like kinase Don3 are both essential for triggering cell separation during cytokinesis. Since Don3 does not contain a Cdc42/Rac interaction and binding domain (CRIB), it is unclear how Cdc42 and Don3 cooperate in the regulation of cytokinesis. To analyse the regulatory network we generated an analogue-sensitive Don3 variant (Don3-as) that allows specific inhibition in vivo. The engineered kinase Don3(M157A) is fully active in vivo and can be specifically inhibited by low concentrations of the ATP-analogue NA-PP1. Inhibition of the Don3-as kinase activity immediately blocked cell separation resulting in the formation of clusters of nonseparated cells. Covalent labelling of cell wall proteins showed that, upon release of inhibition, cytokinesis was resumed instantaneously in all cells. By sequentially activating Don3 and Cdc42 we were able to demonstrate that both proteins act independently of each other and that Don3 activity precedes that of Cdc42. We provide evidence that Don3 and Cdc42 are crucial for the assembly of a contractile actomyosin ring, which is a prerequisite for secondary septum formation. We propose, that Don3 is involved in establishing a landmark, at which the Cdc42-dependent actomyosin ring formation will occur.