The sea urchin egg is a quiescent cell...until fertilization, when the egg is activated. The classic respiratory burst at fertilization is the result of prodigious hydrogen peroxide production, but the mechanism for this synthesis is not known. Here we quantitate the kinetics of hydrogen peroxide synthesis at a single-cell level using an imaging photon detector, showing that 60 nM hydrogen peroxide accumulates within the perivitelline space of each zygote. We find that the NADPH oxidation activity is enriched at the cell surface and is sensitive to a pharmacological inhibitor of NADPH oxidase enzymes. Finally, we show that a sea urchin dual oxidase homolog, Udx1, is responsible for generating the hydrogen peroxide necessary for the physical block to polyspermy. Phylogenetic analysis of the enzymatic modules in Udx1 suggests a potentially conserved role for the dual oxidase family in hydrogen peroxide production and regulation during fertilization.