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 Ca²⁺ concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca²⁺concentration increase is due to the persistent activation of a Ca²⁺ influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca²⁺ store depletion–activated Ca²⁺ entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca²⁺ permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca²⁺ influx through a store depletion–operated pathway and that this drives the exocytotic acrosome reaction.