Previous studies in our laboratories (Miyata, T., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 6132-6136) showed that the structural defect in a hereditarily abnormal plasminogen, plasminogen Tochigi, is due to replacement of Ala by Thr at position 600 from the NH2-terminal end. In the present studies, two abnormal plasminogens, plasminogens Tochigi II and Nagoya, obtained from other family members were analyzed to identify the structural impairment in these molecules. Amino acid sequence analysis of one of the tryptic peptides isolated, respectively, from plasminogens Tochigi II and Nagoya indicated that in both cases, Ala-600 (equivalent to Ala-55 of the chymotrypsin numbering system) had been replaced by Thr. No other substitutions at the active site and substrate-binding site residues, namely, His-57, Asp-102, Ser-195, and Asp-189, were found in the plasmin light chain variants, indicating that all these residues are intact. Moreover, the NH2-terminal heptapeptide sequences of the plasmin light chain variants isolated from plasminogens Tochigi II and Nagoya were identical to the sequence determined for the normal control. These results indicate that the absence of proteolytic activity of both abnormal molecules is due to the same amino acid substitution as that of previously reported plasminogen Tochigi.