Cytokinesis is the process by which the cytoplasm of a eukaryotic cell is divided into two daughter cells, and results from the RhoA-dependent assembly and contraction of an actin–myosin ring at the cell equator. The mitotic spindle, which is formed from astral and central spindle microtubules, contributes to the formation of the actin–myosin ring by sending signals from both the spindle midzone and the astral microtubules to control the localisation of contractile proteins. It is not known, however, how these microtubules work together in human cells to define the division plane. Alisa Piekny and colleagues now address this issue on page 3699. The authors show that astral microtubules restrict the cortical localisation of both the contractile-ring-stabilising protein anillin and RhoA in mitotic cells, whereas the central spindle promotes the accumulation of RhoA in a discrete region and helps to form a tight contractile ring. Therefore, astral and central spindle microtubules independently contribute to the localisation of contractile proteins during cytokinesis. The authors also show that human anillin localises to astral microtubules and can bind to microtubules in vitro, similar to its homologue in Caenorhabditis elegans, and that active RhoA could compete with microtubules for anillin localisation. In addition, they show that anillin is essential for regulating the localisation of proteins at the polar cortex, regardless of changes in astral microtubules. These data therefore describe how the astral pathway contributes to the localisation of contractile proteins, and identify anillin as a potential component of this pathway.