In contrast to most systems in which oocyte activation is triggered by the fertilizing sperm, Sicyonia ingentis oocytes are activated by seawater Mg2+ during spawning. S. ingentis oocytes were spawned into Mg2+-free seawater and microinjected with the fluorescent Ca2+ indicator Fluo-3 to study the effects of added Mg2+ on intracellular Ca2+ levels. The Mg2+ induced a wave of fluorescence across the oocyte that traveled at a speed of 13 ± 3 μm/sec. Extracellular Ca2+ was not required for induction of the wave. Treatment with Ca2+ ionophore in Mg2+-free medium or a localized injection (0.3% oocyte volume) of 3-5 μM Ca2+ also initiated the wave; injection of 250 mM Mg2+ (up to 1.5% oocyte volume) had no effect. Microinjection of 750 μM EGTA (final) suppressed the Mg2+-induced wave, while an identical concentration of EDTA had no inhibitory effect. Subsequent to the initial Mg2+-induced intracellular Ca2+ increase, a second Ca2+ increase was observed at approximately 15 min postspawning; the timing of this second increase appeared to be independent of when the Mg2+-induced wave was initiated, thus an event associated with spawning may be involved. While oocytes in normal seawater were monospermic, those in Mg2+-free seawater were polyspermic, suggesting a role for the Mg2+-induced Ca2+ wave in regulating sperm entry into the oocyte.