Unfertilized and fertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus, injected with the fluorescent probe calcium green dextran (Molecular Probes, Inc.) and monitored with single cell fluorimetry, rarely responded to 10 mM caffeine, but with 20 mM caffeine they produced a transient rise in intracellular calcium. Unfertilized eggs of both species produced a sharp peak approximately 2/3 the height of the normal fertilization peak, with the L. pictus response usually slightly lower and more variable. Image-intensified video microscopy of S. purpuratus eggs showed that this release originated in the center of the egg as well as in the cortex. When caffeine was applied to fertilized eggs within 10-15 min after the fertilization peak, during the period of elevated calcium, the size of the resulting calcium release increased with the elapsed time between the peak and the time of caffeine application. For example, there was no release when caffeine was applied at the height of the peak. When caffeine was washed out after the calcium level returned to baseline, there was a secondary peak (in the absence of caffeine) whose size decreased in proportion to the elapsed time. The source of this secondary calcium release is not known. Caffeine applied after the fertilization peak had returned to baseline produced a transient peak that was not followed by a secondary peak. The caffeine response gradually increased during the cell cycle. Although 10 mM caffeine did not produce a measurable calcium release, light microscopy of fixed and sectioned material showed that fertilized eggs incubated throughout the cell cycle in 7-10 mM caffeine-sea water had greatly reduced numbers of microtubules. Eggs treated at prometaphase with 10 mM caffeine rapidly lost a formed mitotic apparatus by shortening of the spindle and movement of the two centrosomes to the metaphase plate. Within 15 min all the mitotic apparatus-associated vesicles, which are known to sequester calcium, aggregated into two dense spheres of packed membranes located at the sites of the former asters. Microtubules and asters rapidly recovered when caffeine was removed. These observations suggest: (1) that the caffeine-sensitive store contributes to the fertilization calcium release and is an important sink for calcium after the fertilization peak, (2) that the caffeine-sensitive store in unfertilized eggs is located in the central part of the egg as well as in the cortex, and (3) that the caffeine-sensitive store is associated with the membrane system of the mitotic apparatus and may serve as a regulator of microtubule polymerization.