The purpose of the present study was to examine the involvement of caspase 3 on H₂O₂-induced apoptosis and its correlation with the calcium signal. Human semen was obtained from healthy volunteers at the Extremadura Center of Human Assisted Reproduction of Badajoz with local ethical committee approval. Samples were collected after 4-5 days of abstinence, and were allowed to liquefy for 30 min. To determinate caspase 3 activity, stimulated or resting cells were lysated and incubated with caspase 3 substrate (AC-DEVD-AMC), and measured with a fluorescence spectrophotometer with excitation wavelength of 360 nm and emission at 460 nm. Our results show that treatment of spermatozoa with increasing concentrations of H₂O₂ (10 mM-1 mM) resulted in an increase in the caspase 3 activity. Cell stimulation with H₂O₂ for 30 min caused a detectable activation of caspase-3 at 10 mM (120±14 % above control). The effect of H₂O₂ on caspase activation was also time-dependent, reaching a maximal caspase activity after 60 min of stimulation at 10 mM with a increase of 130.6 ± 16.9 % above control. The stimulatory effect of H₂O₂ on caspase activation was significantly reduced by pretreatment of cells for 30 min with either the intracellular chelator dimethyl BAPTA or Ru360, a blocker of Ca²⁺ uptake into mitochondria. Cell treatment for 90 min with 100 mM z-LEHD-FMK (an inhibitor of caspase-9) abolished H₂O₂-induced caspase-3 activation. Our results suggest that H₂O₂-induced mitochondrial apoptosis in human spermatozoa is dependent of calcium signalling. This work was supported by Laboratorios SERONO, S.A.