Sea urchin mitotic apparatuses (MAs) were isolated in a microtubule stabilizing buffer that contained detergent. These isolated MAs contain a calcium and calmodulin-dependent protein kinase that phosphorylates one specific MA-associated endogenous substrate with a relative molecular mass of 62 kd. No protein phosphorylation occurs in the presence of calcium or magnesium ion alone, or when magnesium ion is combined with 10 microM cyclic AMP or cyclic GMP. Because in vivo labeling studies showed that the 62 kd protein was also phosphorylated in living cells during mitosis, the effect of protein phosphorylation on MA stability was also studied. When isolated MAs were incubated under conditions that resulted in phosphorylation of the 62 kd protein, substantial depolymerization of MA microtubules occurred within 10 min. MAs incubated under similar conditions but in the absence of 62 kd phosphorylation lost many fewer microtubules and were stable for up to 30 min. The results are discussed with respect to a model for mitosis in which the specific role of protein phosphorylation in the events of anaphase is addressed.