The complete disulfide loop structure of human α2-HS glycoprotein has been elucidated. α2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of α2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665–1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403–4407), whereas the light chain corresponds to the β-chain of α2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966–4971). Twelve half-cystine residues are present in the α2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of α2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4–19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in α2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that α2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of α2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.