The 228 amino acid primary sequence of proteinase-3 (PR3) was studied for antigenic epitopes, which react with IgG Ab in sera of patients with Wegener's granulomatosis, by synthesizing overlapping 7 mers of PR3 linear sequence on pins. Main regions of linear sequence reactivity included GHEAQPH (at residues 4-10), QPHSRPY (8-14), TQEPTQQ (65-71), ATVQLPQ (108-114), QLPQQDQ (111-117), PVPHGTQ (118-124), RVGAHDP (132-138), FCRPHNI (154-160), PRRKAGI (165-171), FGDSGGP (173-179), and IDSFVIW (189-195). All of these regions were surface accessible and seemed to be on the outside of the molecule. By substituting a glycine or an alanine for each residue within each reactive epitope, we identified the arginine at 12, valine at 119, proline at 120, histidine at 121, arginine at 132, alanine at 135, histidine at 136, and glycine at 178 as major immunodominant single residues that contribute to antigenic determinants. Visualization of these reactive regions on a three-dimensional computer-generated carbon PR3 trace diagram showed that one aspect of the molecule on the inferolateral surface comprised most of the antigenic regions. Two peptides, ATVQLPQ and RVGAHDP, when preincubated with Wegener's granulomatosis sera, produced almost complete inhibition of Wegener's granulomatosis IgG Abs either binding to PR3 on the ELISA plate or by reacting with polymorphonuclear leukocyte azurophilic granules in immunofluorescence assays for cANCA. FGDSGGP (at residues 173-179), one epitope that was recognized by Wegener's patient sera Abs, included part of the active catalytic triad for PR3. Rabbit Abs against the PR3 peptides ATVQLPQ or RVGAHDP showed a cANCA cytoplasmic granular pattern of immunofluorescence staining when they were incubated with polymorphonuclear leukocytes. These findings indicate that it is possible to identify various antigenic sites on PR3 by using overlapping 7 mers of portions of the solvent, accessible linear sequence.