The relationship between progression through anaphase and furrow establishment was investigated in PtK1 cells using the anti-mitotic agent Nocodazole to arrest cells at different points in anaphase. The capacity of cells to furrow was compared to the kinetochore-kinetochore separation attained at the time of arrest. For the stages of anaphase examined, furrowing capacity increased directly with kinetochore-kinetochore separation until complete furrows were formed after kinetochore-kinetochore separations of 14 micron or more were reached. Furrow establishment thus occurs during a definite interval during anaphase in PtK1 cells. Results from electron microscopy of both Nocodazole-treated and control cells suggest that a population of astral microtubules may be important for furrow establishment.