In order to elucidate the role of spermosin (a novel sperm trypsin-like protease) in ascidian fertilization, we developed an antibody against the ascidian spermosin: the antibody appears specific to the spermosin but not to the acrosin. By Western blot analysis, spermosin was found to be expressed just before and during the spawning season. Immunocytochemical studies have shown that spermosin is localized on the sperm head. It was found that the spermosin was released from the sperm during the sperm reaction, and the anti-spermosin antibody, but not control antibody, inhibited the fertilization in a concentration-dependent manner. These results indicate that spermosin plays an essential role in ascidian fertilization.