The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca² -free medium and in the presence of Ca² channel antagonists. We also examined the GnRH effect on the intracellular free Ca² concentration ([Ca² ]_i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca² was present in the medium. In Ca² -free medium or in the presence of 400 nM nifedipine, 80 μM diltiazem, or 50 μM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca² ]_i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca² ]_i was fast and transient, from a basal [Ca² ]_i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca² ]_i was entirely due to a Ca² influx from the extracellular medium because the increase in [Ca² ]_i was blocked by the Ca² chelator EGTA and by the Ca² channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca² influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca² influx but inhibited the progesterone-activated Ca² influx. Finally, the GnRH-induced Ca² influx was blocked by two specific GnRH antagonists, Ac-D-Nal¹-Cl-D-Phe²-3-Pyr-D-Ala³-Arg⁵-D-Glu(AA)⁶-GnRH and Ac-^3,4-dehydro-Pro¹,-p-fluoro-D-Phe², D-Trp^3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca² ]_i through T-type, voltage-operated calcium channels.