Although the Acrosome Reaction (AR) is a fundamental exocytic event for fertilization, the signaling pathway involved in this process is not completely understood. The physiological induction of the AR triggers a biphasic Ca²⁺ influx; however, the molecular entities involved in this process are not clearly defined. A model of the signaling pathway during the AR that implies the participation of the Exchange Protein directly Activated by cAMP (Epac) has been proposed. We speculate that Epac might activate the enzyme (CD38) that catalyzes the production of nicotinic acid adenine dinucleotide phosphate (NAADP), which in turn activates the intracellular Two Pore Channels (TPC), releasing a small amount of Ca²⁺ that would convey the signal to other channels (IP₃R and RyR) releasing more Ca²⁺, and amplifying the response. In the present work we employ the EPAC specific cAMP analogue (8-pCPT-2'-O-Me-cAMP) to trigger the Ca²⁺ rise in the presence or absence of specific channel inhibitors to confirm or rule out the participation of these components in the proposed signaling pathway. We would characterize the process by 1) Measuring the intracellular Ca²⁺ change in sperm loaded with a fluorescent Ca²⁺ indicator and 2) Evaluating the percentage of sperm that undergo the AR in different conditions. Our preliminary results suggest that the TPC and IP₃R are implicated in the signaling pathway of the AR trigger by Epac.