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To explore the biological characteristics of the recombinant zona pellucida 3 (ZP3) peptides of rhuZP3a^22∼176 and rhuZP3b^177∼348, we examined whether rhuZP3a^22∼176 or rhuZP3b^177∼348 trigger the acrosome reaction (AR) of human spermatozoa and we investigated the underlying mechanism. The assessment of AR was performed using chlortetracycline staining. The intracellular free calcium concentration ([Ca²⁺]i) in Fura‐2/AM‐loaded human sperm was monitored with a spectrofluorophotometer. We found that rhuZP3a^22∼176 and rhuZP3b^177∼348 were capable of eliciting AR at different concentrations. With the addition of either peptide, the [Ca²⁺]i level was raised to a peak with or without a plateau. The AR could be inhibited by pertussis toxin (PTX), EGTA, and pimozide (a T‐type calcium channel blocker), whereas verapamil was less effective in this regard. The results of the present study …