Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vκ1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vκ5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vκ1c-expressing hybridomas. Intriguingly, all 10 Vκ1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated Vκ1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.