Progesterone production by the corpus luteum is a process vital for reproduction. In humans its secretion is stimulated by the placental hormone human chorionic gonadotropin (hCG), and this stimulatory action can also be observed in cultured human luteinized granulosa cells (GCs). We now provide evidence that opening of a Ca(2+)-activated K(+) channel, the BK(Ca), is crucially involved in this process. Immunohistochemistry and RT-PCR revealed the presence of the pore-forming alpha-subunit in human luteinized GCs and in luteal cells of human, macaque, and rat, implying that BK(Ca) channels are important throughout species. Blocking of BK(Ca) channels by iberiotoxin attenuated hCG-induced progesterone secretion. The inhibitory action of iberiotoxin suggests that BK(Ca) channels are activated in the course of hCG-induced steroidogenesis. In search of physiological activators we used an electrophysiological approach and could preclude a direct regulation of channel activity by hCG or GC-derived steroids (progesterone and 17beta-estradiol). Instead, the peptide hormone oxytocin and an acetylcholine (ACh) agonist, carbachol, evoked transient BK(Ca) currents and membrane hyperpolarization. These two molecules are both secreted by GCs and act via raised intracellular Ca(2+) levels. The release of oxytocin is stimulated by hCG, and a similar mechanism is likely in the case of ACh. We conclude that BK(Ca) channel activity in GCs is mediated by components of the intraovarian signaling system, thereby interlinking a systemic hormonal and a local neuroendocrine system in control of steroidogenesis.