To determine the role of calcium signaling on apoptosis evoked by the reactive oxygen species H 2 O 2 and by the physiological agonist P in human ejaculated spermatozoa.Laboratory study.Center for assisted human reproduction in a hospital in Spain.Forty-five healthy volunteers.Spermatozoa were treated with increasing concentrations of hydrogen peroxide (H 2 O 2 ; 10 μM, 100 μM, and 1 mM) or with 20 μM of P for 5–120 minutes.Activation of caspase-3 and -9 as well as phosphatidylserine externalization were examined in human ejaculated spermatozoa by fluorescence methods.Hydrogen peroxide and P induced activation of caspase-3 and -9. In addition, the effect of H 2 O 2 and P was time dependent. Dimethyl-1,2-bis (aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid loading was able to inhibit H 2 O 2 - and P-induced caspase-3 activation and phosphatidylserine externalization. Pretreatment of spermatozoa with Ru360, to block the calcium uptake into mitochondria, also was able to decrease the activation of caspase-3 and phosphatidylserine exposure that was stimulated by either H 2 O 2 or P.These findings suggest that H 2 O 2 - and P-induced mitochondrial apoptosis is dependent on calcium signaling.