AbstractQuinacrine, an acridine derivative which competitively binds to ATP binding sites, has been used to study the role of ATP requiring molecules in microtubule organization in mitotic PtK1cells. Brief treatments of metaphase cells with concentrations of quinacrine ranging from 2 to 10 μM decreased spindle length and birefringence in a concentration‐dependent manner. With either increasing quinacrine concentrations or duration of treatment, metaphase cells demonstrated a specific reorganization of spindle microtubules. Both polarization and electron microscopy showed a substantial loss of non‐kinetochore spindle microtubules with an increase in astral microtubules: this was particularly evident in the region adjacent to the spindle domain. Addition of millimolar concentrations of dinitrophenol to quinacrine‐containing medium did not potentiate the response of metaphase cells to quinacrine treatment. Time‐lapse video analysis demonstrated that the astral microtubules are the result of reorganization of spindle microtubules. These data suggest that functional ATP binding sites are required to maintain stable interactions between microtubules and that these interactions are responsible for maintaining the bowed configuration of non‐kinetochore spindle microtubules which are under compression at