|Abstract||Sperm-specific phospholipase C zeta (PLC?) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLC? C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLC?. Microinjection of cRNA or recombinant protein corresponding to PLC?I489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLC?I489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLC?I489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLC?. Our findings highlight the importance of PLC? at fertilisation and the vital role of the C2 domain in PLC? function, possibly due to its novel binding characteristics.