Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M.W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-γ-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.