We have previously described a phospholipase C (PLC) activity in mammalian sperm cytosolic extracts. Here we have examined the Ca²⁺ dependency of the enzyme, whether there is enough in a single sperm to account for Ca²⁺ release at fertilization, and finally where in the egg is the phosphatidyl 4,5-bisphosphate, the substrate for the enzyme. As for all PLCs examined so far in vitro, we found that the boar sperm PLC activity was Ca²⁺ dependent. Specific activity increased when free Ca²⁺ levels were micromolar. However, even at nanomolar free Ca²⁺ concentration the boar sperm PLC activity was considerable, being two orders of magnitude greater than PLC activities in other tissues. We calculated that PLC activity of a single boar sperm in a mammalian egg is enough to generate 400 nM inositol 1,4,5-trisphosphate (InsP₃) in 1 min, which may be sufficient to account for the observed Ca²⁺ changes in an egg at fertilization. We fractionated sea urchin egg homogenate and examined the ability of boar sperm extract to generate InsP₃ from these fractions. The sperm PLC activity triggered InsP₃ production from a PIP₂-enriched nonmicrosomal egg compartment that contained yolk platelets. We propose that this sperm PLC activity, which is active at nanomolar Ca²⁺ levels and hydrolyzes PIP₂ from intracellular membranes, could be involved in the Ca²⁺ changes observed at fertilization.