Progesterone is capable of initiating the mammalian sperm acrosome reaction in vitro and is a putative initiator of this essential fertilization event in vivo. Our previous work has suggested that progesterone initiates the human sperm acrosome reaction, at least in part, by activating a unique steroid receptor/Cl- channel resembling a gamma-aminobutyric acidA receptor/Cl- channel (gamma-aminobutyric acidA-like receptor/Cl- channel). Here, the fluorescent intracellular Cl- probe, 6-methoxy-N-ethylquinolinium, was used to detect qualitative changes in sperm cytosolic Cl-. We demonstrate that progesterone can mediate a rapid transient decrease of human sperm cytosolic Cl- inhibitable by the gamma-aminobutyric acidA receptor/Cl- channel antagonists picrotoxin and (+)-bicuculline (which also inhibit the acrosome reaction). These results support the involvement of a gamma-aminobutyric acidA-like receptor/Cl- channel in the P-mediated human acrosome reaction and are the first to demonstrate that a rapid Cl- efflux plays a role in that event.