AbstractProteinase 3 (PR3) is a neutrophil‐derived serine proteinase localized within cytoplasmic granules which can be released upon activation. PR3 is exposed at the neutrophil plasma membrane where it can mediate proinflammatory effects. Moreover, PR3 membrane expression is of special relevance in patients with Wegener's granulomatosis, a systemic vasculitis presenting anticytoplasmic neutrophil autoantibodies (ANCA) against PR3, which can bind to PR3 expressed at the surface of neutrophils and amplify their activation state. Therefore, it is of special relevance to unravel the molecular mechanisms governing its association with the membrane to be able to modulate it. To this end, we performed molecular dynamics (MD) simulations of PR3 with the implicit membrane model IMM1‐GC to identify its interfacial binding site (IBS). Both the energies and structures resulting from the MD suggest that PR3 associates strongly with anionic membranes. We observe a unique IBS consisting of five basic (R177, R186A, R186B, K187, R222) and six hydrophobic (F165, F166, F224, L223, F184, W218) amino acids. The basic residues provide the driving force to orient PR3 at the membrane surface, so that the hydrophobic residues can anchor into the hydrocarbon region. Energy decomposition and in silico mutations show that only a few residues account for the membrane association. Similar calculations with HNE suggest a different membrane‐binding mechanism. Our results agree with previous experimental observations and this work predicts, for the first time, the structural determinants of the binding of PR3 to membranes. Proteins 2008. © 2007 Wiley‐Liss, Inc.