Spermatozoa bind to the vitelline coat in the ascidians and many other animals. The binding of sperm in Halocynthia roretzi is mediated by a sperm α-l-fucosidase and complementary-l-fucosyl residues of glycoproteins in the vitelline coat. cDNA clones for α-l-fucosidase were isolated from growing testis mRNA. It contained a 1398 bp full-length cDNA insert (HrFuc'ase) that encoded the 466 amino acid residues of H. roretzi sperm α-l-fucosidase. A putative signal peptide of 21 amino acid residues proceeded the sequence for the mature protein (M.W. 52.4 kDa). The coding sequence for HrFuc'ase showed 47.7% sequence identity to the human liver fucosidase sequence. The polyclonal antibody was prepared against a lacZ-HrFuc'ase fusion protein expressed in E. coli. The antibody crossed to a 54 kDa protein in sperm on western blotting and inhibited fertilization in a dose dependent manner. These data suggest that sperm-egg binding is mediated by the sperm α-l-fucosidase, HrFuc'ase in the ascidian, H. roretzi.