The role of calmodulin in fertilisation events 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 calcium oscillations induced by fertilisation by using the Ca2+ indicator, fluo-3/AM, or on pronucleus formation 4 h later by using the nucleic acid stain, Syto-15. When the calmidazolium treatment was applied to one or the other gamete before insemination, the fertilisation process was affected only when spermatozoa were treated: most of the oocytes were partially fertilised as demonstrated by the profile of Ca2+ oscillations and the presence of polar bodies with no typical male and female pronuclei. When the treatment was applied during insemination, more than half the oocytes were unfertilised and only a few were partially fertilised. These results demonstrate that: (1) the calmodulin-dependent events taking place in spermatozoa before insemination appear essential at least for regular Ca2+ oscillations and for pronucleus formation; (2) the inhibition of calmodulin by calmidazolium applied to metaphase II oocytes before insemination has no major impact on their fertilising ability; and (3) at the time of gamete fusion calmodulin, either from the oocyte or from the spermatozoon, is essential for fertilisation to occur.