In human spermatozoa, Ca² entry is stimulated by progesterone or prostaglandin E₁ (PGE₁). The regulation of cation currents by progestins involves sigma receptors, and sigma binding sites are abundant in testis. We examined the effects of sigma ligands on human spermatozoa. Ca² entry induced by progesterone or PGE₁ was not altered by the sigma ligands haloperidol and ditolylguanidine. However, the steroidal sigma ligands RU 3117 and RU 1968 had distinct effects. Stimulation by RU 3117 resulted in activation and homologous desensitization of the sperm progesterone receptor but not of the PGE₁ receptor. Because haloperidol and ditolylguanidine did not affect RU 3117 and progesterone actions in spermatozoa, we conclude that sigma receptors are not involved. However, RU 1968 potently inhibited both the progesterone- and PGE₁-induced Ca² entry and acrosome reaction. At higher concentrations, RU 1968 also inhibited hormonal Ca² signaling in fibroblasts. Despite suppression of Ca² mobilization, inhibition of phospholipase C by RU 1968 was not observed. Furthermore, RU 1968 did not impair the binding of inositol-1,4,5-trisphosphate to its endoplasmic reticulum receptor. Because RU 1968 preferentially inhibits signaling pathways in spermatozoa, the future development of more selective drugs structurally related to RU 1968 may be a novel approach for pharmacological contraception.