The role of calmodulin in fertilisationevents was examined in a zona-free mouse system by using a selective calmodulin inhibitor,calmidazolium (1 μM). The effects of this antagonist were studied either on the ooplasmic calciumoscillations induced by fertilisation by using the Ca2+ indicator, fluo-3/AM, or onpronucleus formation 4 h later by using the nucleic acid stain, Syto-15. When the calmidazoliumtreatment was applied to one or the other gamete before insemination, the fertilisation process wasaffected only when spermatozoa were treated: most of the oocytes were partially fertilised asdemonstrated by the profile of Ca2+ oscillations and the presence of polar bodies with notypical male and female pronuclei. When the treatment was applied during insemination, more than halfthe oocytes were unfertilised and only a few were partially fertilised. These results demonstratethat: (1) the calmodulin-dependent events taking place in spermatozoa before insemination appearessential at least for regular Ca2+ oscillations and for pronucleus formation; (2) theinhibition of calmodulin by calmidazolium applied to metaphase II oocytes before insemination has nomajor impact on their fertilising ability; and (3) at the time of gamete fusion calmodulin, eitherfrom the oocyte or from the spermatozoon, is essential for fertilisation to occur.