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We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al . (1984), J. Biol. Chem. 259 , 2900–2904; Sawada et al . (1984), Dev. Biol. 105 , 246–249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130–388) and a long light chain designated L1 (residues 23–129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97–129). The L1 chain contains a proline-rich region, designated L1(ΔL2) which is lacking in L2. Investigation with the glutathione-S -transferase (GST)–spermosin-light-chain fusion proteins, including GST–L1, GST–L2, and GST–L1(ΔL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization. [ABSTRACT FROM AUTHOR]