In this study the effect of the sequential exposure of spermatozoa to progesterone and gonadotrophin-releasing hormone (GnRH) upon zona binding and the intracellular free Ca2+ concentration was evaluated. Sperm aliquots were treated as follows: (a) 0.7 μmol l-1 progesterone or 0.1% DMSO (progesterone solvent) followed by 50 nmol l-1 of GnRH; (b) 50 nmol l-1 of GnRH or distilled water (GnRH solvent) followed by 0.7 μmol l-1 of progesterone. Additional aliquots were incubated with DMSO or distilled water (controls) and with 0.7 μmol l-1 of progesterone or 50 nmol l-1 of GnRH. All treatments were for 5 min. Motile spermatozoa were incubated in modified Tyrode's medium, at 37°C, 5% CO2, 10 × 106 spermatozoa ml-1, for 4.5 h. Intracellular Ca2+ concentration and sperm-zona binding was evaluated using fura 2 and the hemizona assay, respectively. GnRH and progesterone increased sperm-zona binding and the Ca2+ concentration. Regarding zona binding, the effect of GnRH was significantly greater when the spermatozoa had been previously treated with progesterone (progesterone → GnRH = 185 ± 116 zona-bound spermatozoa versus DMSO → GnRH = 99 ± 15, P < 0.001). On the other hand, previous treatment with GnRH did not modify their subsequent response to progesterone (GnRH → progesterone = 114 ± 19 zona-bound spermatozoa versus distilled water → progesterone = 108 ± 22, NS). The results regarding intracellular Ca2+ showed a similar pattern. These findings suggest a priming effect of progesterone upon a GnRH-induced increase in sperm-zona binding and intracellular Ca2+.