A novel strategy in cancer therapy is the induction of mitotic cell death by the pharmacological abrogation of cell cycle checkpoints. UCN-01 is such a compound that overrides the G2 cell cycle arrest induced by DNA damage and forces cells into a deleterious mitosis. The molecular pathways leading to mitotic cell death are largely unknown although recent evidence indicates that mitotic cell death represents a special case of apoptosis. Here, we demonstrate that the mitotic spindle checkpoint is activated upon chemotherapeutic treatment with topoisomerase II poisons and UCN-01. Cells that are forced to enter mitosis in the presence of topoisomerase inhibition arrest transiently in a prometaphase like state. By using a novel pharmacological inhibitor of the spindle checkpoint and spindle checkpoint-deficient cells we show that the spindle checkpoint function is required for the mitotic arrest and, most importantly, for efficient induction of mitotic cell death. Thus, our results demonstrate that the mitotic spindle checkpoint is an important determinant for the outcome of a chemotherapy based on the induction of mitotic cell death. Its frequent inactivation in human cancer might contribute to the observed resistance of tumor cells to these chemotherapeutic drugs.