Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.