Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.