A trypsin inhibitor from hemolymph of the solitary ascidian, Halocynthia roretzi, has been reported to be present in two forms, ATI-I and ATI-II. ATI-I consists of a single polypeptide chain with a unique sequence of 55 amino acid residues, while ATI-II has two chains that seem to be derived from ATI-I by cleavage at the Lys16-Met17 bond [Kumazaki, T., Hoshiba, N., Yokosawa, H., and Ishii, S. (1990) J. Biochem. 107, 409-413]. ATI-II (as modified inhibitor) was proved to be produced by incubation of ATI-I (as virgin inhibitor) with a catalytic amount of bovine trypsin. The tryptic hydrolysis at the Lys16-Met17 bond in virgin inhibitor showed a maximum velocity at around pH 3.5. On the other hand, acid treatment of a complex prepared by mixing equimolar quantities of trypsin and the the modified inhibitor yielded free trypsin and the virgin inhibitor. The results of chemical analyses indicated that the Lys16-Met17 bond that had been cleaved in ATI-II was resynthesized by the acid treatment. These findings strongly suggest that the Lys16-Met17 bond is the reactive site of ATI for trypsin.