Transient elevations in intracellular free Ca 2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca 2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca 2 into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar ( Echinaracnius parma) embryos was microinjected with Ca 2+ buffers, Ca 2+ solutions, or Ca 2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca 2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca 2+-dependent steps in NEB and initiation of mitosis. Injection of 50 μM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 μM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 μM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca 2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca 2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca 2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 μM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 μM CaCl 2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca 2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 μg/ml ryanodine and 500 μg/ml TMB-8. These results indicate that a transient elevation of intracellular Ca 2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset. These findings support the existence of a cell cycle clock, and further support the hypothesis that Ca 2+ transients may be used to coordinate and synchronize the biochemical pathways required for NEB, anaphase onset, and mitosis.