In human spermatozoa, progesterone (P₄) induces a depolarization of the plasma membrane, a rapid calcium (Ca² ) influx, and a chloride efflux. The sodium ion (Na ) was partly responsible for the P₄-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P₄ induced a Na influx and whether voltage-operated channels were involved in Na and/or Ca² entries. We found that 10 μM P₄ significantly increased intracellular Na concentration from 17.8 ± 2.0 mM to 27.2 ± 1.6 mM (P < 0.001). Prior incubation of spermatozoa with 10 μM flunarizine, a Na and Ca² voltage-dependent channel blocker, inhibited the sodium influx induced by 10 μM P₄ by 84.6 ± 15.4%. The Ca² influx induced by 10 μM P₄ was also significantly inhibited in a Na -containing medium by 10 μM flunarizine or 10 μM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca² influx induced by 10 μM P₄ in spermatozoa incubated in Na -depleted medium. The P₄-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na -containing medium as compared to Na -depleted medium. These data demonstrate that P₄ stimulates a Na influx that could be involved in the AR completion. They also suggest that voltage-dependent Na and Ca² channels are implicated in P₄-mediated signaling pathway in human spermatozoa.