The mitotic apparatus of sea urchin embryos was isolated using a polyethylene glycol (PEG)/EGTA-medium. Such a procedure preserves the birefringence and the Ca2+ lability of the isolated mitotic apparatus. The method of isolation gives good preservation of the microtubules and of the intracellular Ca2+-transport system as visualized by a monoclonal antibody to a 46-kDa protein. Triple fluorescence studies allow a comparison of the relative locations of microtubules, Ca2+-sequestering membranes and chromatin (by Hoechst 33342) in the mitotic apparatus. We find that the Ca2+-sequestering membranes are concentrated mainly in the centers of the asters and do not follow the distribution of microtubules in the mitotic apparatus. Regulation of microtubules by Ca2+ may not depend on immediate contiguity of microtubules and the Ca2+-regulating sites.