Mammalian spermatozoa become competent for fusion with oocytes while traveling through the female reproductive tract and the oocyte's extracellular investments. Recent studies highlighted the molecular mechanism of the sperm's interactions with the zona pellucida (ZP), the extracellular coat surrounding the oocyte. Fertilizing spermatozoa initiate the sperm acrosome reaction (AR), essential for zona penetration and fusion with the oocyte plasma membrane, before they reach the ZP. However, the exact condition of spermatozoa that leads to successful penetration of the ZP remains unknown. We performed microscopic observations of in vitro fertilization with genetically (EGFP) and chemically (antibody and lectin) labeled spermatozoa to monitor the progression of the AR. Spermatozoa exhibiting EGFP(-)/PNA(+) prior to binding to the ZP initiated zona penetration. This result suggests that spermatozoa that have undergone the AR are still capable of binding and penetrating the ZP.