INTRODUCTIONThe centrosome is a cell's primary microtubule-organizing center. In most mammalian cells, the centrosome is composed of a pair of centrioles and surrounding pericentriolar material. The centrosome is duplicated exactly once per cell cycle such that at the onset of mitosis, a cell has two centrosomes, which serve as poles of the mitotic spindle. During cytokinesis, one centrosome is segregated to each daughter cell. This protocol describes the isolation of centrosomes from asynchronous cells, and thus the purified material will consist primarily of interphase centrosomes. Isolated centrosomes can be used in a variety of assays, including studies of microtubule function and the identification of centrosome-associated proteins and their interactions.