The nuclear-centrosome complex was isolated from interphase Chinese hamster ovary (CHO) cells, and, with exogenous brain tubulin as a source of subunits, the centrosome, while attached to the nucleus, was demonstrated to nucleate microtubule formation in vitro. We attempted to quantitate the nucleating activity in order to compare the activity of mitotic and interphase centrosomes. However, the proximity of the nucleus hindered these attempts, and efforts to chemically or mechanically remove the centrosome led to diminished nucleating activity. Therefore, the nuclear-centrosome complex was dissociated biologically through use of the cytochalasin B procedure for enucleation of cells. Cytoplasts were prepared that retained the centrosome. Lysis of the cytoplasts released free centrosomes that could nucleate microtubules in vitro. The nucleating activities of interphase and mitotic centrosomes were compared. In addition, through the use of whole-mount electron microscopy, the configuration of the centrioles was analyzed and the number of microtubules nucleated was determined as a function of the centriole cycle. Nucleating activity did no change discernibly throughout interphase but increased approximately fivefold at the transition to mitosis. Thus, we conclude that the nucleating activity of the centrosome is relatively independent of the centriole cycle but coupled to the mitotic cycle.