The signal transduction pathways involved in the progesterone (P₄)-initiated mammalian sperm acrosome reaction (AR) are not fully understood. To investigate the role of the protein kinase A (PKA) pathway in the P₄-initiated AR, we probed this pathway by pretreating capacitated human sperm with reagents designed to either inhibit PKA activation or disrupt PKA/A kinase anchoring protein (AKAP) interactions. Preincubation with the stearated (membrane permeable) PKA inhibitor, PKI α 5-24 (S-PKI α 5-24), significantly inhibited the P₄-initiated AR at 10 μM as compared to stearated control peptide. In contrast, preincubation with 100 μM nonstearated PKI α 5-24 did not significantly inhibit versus solvent control. Preincubation with the PKA inhibitor Rp-8-Br-cAMP at 500 μM and 150 μM significantly inhibited the P₄-initiated AR versus 8-Br-cAMP and versus solvent. Preincubation with the anchoring inhibitory peptide S-Ht-31 significantly stimulated the P₄-initiated AR at 10, 3, and 1 μM versus inactive control peptide. The stimulation of the P₄-initiated AR by 3 μM S-Ht31 was significantly inhibited by the addition of 30 μM S-PKI α 5-24 prior to the addition of S-Ht31. Preincubation with S-PKI α 5-24 (30 μM) partially inhibited the ionomycin (50 μM)-initiated AR. A role for PKA in the P₄-initiated AR may exist both upstream and downstream of Ca² entry. Our studies present the first evidence for the participation of PKA in the P₄-initiated AR and also suggest that AKAPs are involved in the PKA-mediated events.