The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.