A soluble phospholipase C (PLC) from boar sperm generates InsP₃and hence causes Ca²⁺ release when added to sea urchin egg homogenate. This PLC activity is associated with the ability of sperm extracts to cause Ca²⁺ oscillations in mammalian eggs following fractionation. A sperm PLC may, therefore, be responsible for causing the observed Ca²⁺ oscillations at fertilization. In the present study we have further characterized this boar sperm PLC activity using sea urchin egg homogenate. Consistent with a sperm PLC acting on egg PtdIns(4,5)P₂, the ability of sperm extracts to release Ca²⁺ was blocked by preincubation with the PLC inhibitor U73122 or by the addition of neomycin to the homogenate. The Ca²⁺-releasing activity was also detectable in sperm from other species and in whole testis extracts. However, activity was not observed in extracts from other tissues. Moreover recombinant PLCβ1, -γ1, -γ2, -δ1, all of which had higher specific activities than boar sperm extracts, were not able to release Ca²⁺ in the sea urchin egg homogenate. In addition these PLCs were not able to cause Ca²⁺ oscillations following microinjection into mouse eggs. These results imply that the sperm PLC possesses distinct properties that allow it to hydrolyse PtdIns(4,5)P₂in eggs.