Nocodazole, the rapidly-reversible inhibitor of microtubule polymerization, has been used as a reagent to produce large numbers of mitotic mammalian cells at all stages of cell division. Mitotic cells are accumulated by incubation of cultures with 0.04 μg/ml of Nocodazole. Arrested cells are then harvested and resuspended in fresh medium where they progress through mitosis. HeLa, WI38, L929 and CHO cells proceed through cell division in a semi-synchronous manner following the removal of the drug. Nocodazole has very little effect on interphase metabolism, and following drug release, cells return to a normal cell cycle. Synchronization protocols and yields are presented.