The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G‐proteins, phospholipase A₂ (PLA₂), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPγs (GTPγs, a mimetic of G‐protein activation), arachidonic acid (AA, product of PLA₂ action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPβS (GDP, an inhibitor of G‐protein activation), ONO RS‐82 (ONO, an inhibitor of PLA₂), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA₂, and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15‐min incubation with GTPγs, AA, or PMA, respectively. But GTPγs or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15‐min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G‐proteins, that in turn activate PLA₂ and PLC simultaneously, that finally promote acrosomal exocytosis. Mol. Reprod. Dev. 70: 58–63, 2004. © 2004 Wiley‐Liss, Inc.