Proteinase3 (PR3) and human neutrophil elastase (HNE) are homologous proteases from the polymorphonuclear neutrophils and have been thought for a long time to have close enzymatic specificity. We have used molecular dynamics simulations to investigate and compare the interactions between different peptides and the two enzymes. The important role played especially by the C-terminal part of the peptides is confirmed. We provide a map of the subsites of PR3 and a description of the interaction scheme for six ligands. The main difference between HNE and PR3 concerns S2, S1', S2', and S3'. The recognition subsites in PR3 are interconnected; in particular, Lys99 participates to a hydrophobic (S4) and a polar (S2) pocket. On the basis of the simulations, we suggest that VADVKDR is a highly specific sequence for PR3; enzymatic assays confirm that it is cleaved by PR3 with a high specificity constant (k(cat)/K(m) = 3,400,000 M(-1) s(-1)) and not by HNE.