The three components of clupein (YI, YII and Z) were hydrolyzed by subtilisin Carlsberg and subtilisin Novo. Subtilisin Novo hydrolyzed all three components much faster than did subtilisin Carlsberg, but components YII and Z were hydrolyzed with the same initial rate and faster than component YI. The primary bonds cleaved in component YII were Ser22-Arg23 followed by Ala8-Ser9. Ser21-Arg22 followed by Ala9-Ser10 were the primary bonds cleaved in clupein-Z. It was further demonstrated that nitrated subtilisin Carlsberg had become more specific for cleavage of the Ser-Arg bond compared with the unmodified enzyme. With clupein YI as the substrate, the peptide pattern obtained was too complicated for a particular bond to be identified as the primary attacking point for the two unmodified enzymes. Nitrated subtilisin Carlsberg attacked initially the bonds Arg5-Ser6 and Ser6-Ser7.