Zona pellucida protein 3, a protein of the egg’s extracellular matrix, and progesterone secreted by granulosa cells surrounding the oocyte are regarded as physiological stimuli of sperm acrosome reaction. Signal transduction steps initiated by both stimuli result in influx of Ca²⁺ from the extracellular space. Herein, we propose a role for prostaglandin (PG) E as a physiological inducer of Ca²⁺ influx and acrosome reaction in human spermatozoa. PGE₁ specifically binds to human sperm membranes (K_d = 20.4 nM; B_max = 88 fmol/mg protein) and induces a pertussis toxin-insensitive, transient increase in intracellular Ca²⁺ concentrations, which can be blocked by μM concentrations of La³⁺, Gd³⁺, and Zn²⁺. The kinetic profile was similar to that observed after progesterone challenge. Sequential application of both agonists did not lead to cross-desensitization. E prostaglandins were found to be the only prostanoids with agonistic properties (EC₅₀ values for PGE₁ and PGE₂: <10 nM and 300 nM, respectively). Pharmacological characteristics were not compatible with those of cloned prostanoid receptors indicating the expression of a distinct membrane receptor. Activation of the sperm E prostanoid receptor stimulates incorporation of [α-³²P]GTP azidoanilide into immunoprecipitated Gα_q/11 subunits. Thus, in human sperm, PG induces Ca²⁺ influx and acrosome reaction via a G_q/11-coupled E prostanoid receptor. The block of PGE₁-induced Ca²⁺ transients and acrosome reaction by physiological Zn²⁺ concentrations highlights a role of Zn²⁺ as an endogenous Ca²⁺ channel blocker present in seminal plasma protecting sperm from premature PGE₁-evoked increases in intracellular Ca²⁺ concentrations.