Three isotrypsins from digestive gland of Penaeus vannamei were purified and characterized by molecular, biochemical and kinetic parameters. Purified isotrypsins A, B, and C are glycoproteins with molecular masses between 30.2 and 32.9 kDa, and, therefore similar to other trypsins. The isoelectric points are anionic and different among the three isotrypsins: pH 3.5 for isotrypsin A, pH 3.0 for isotrypsin B, and pH 4.5 for isotrypsin C. Differences in the NH(2)-terminal amino acid sequences allowed us to define three different protein entities that match isotrypsins previously deduced by cDNA. Isoform C has higher physiological efficiency and specific activity, lower K(m), and requires higher concentrations of Ca(+2) to reach the same activity as the other two isotrypsins.