To investigate the origin of disease-associated IgM autoantibodies (AAb), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (mev) mice with natural autoantibodies (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from mev mice, at the terminal stage of systemic autoimmune disease, and from mitogen-stimulated C57/BL6 mice. These were randomly selected for VH J558 gene expression (aberrantly expressed in mev mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmutated germline VH genes. Disease-associated AAb from mev mice were predominantly encoded by the J558 subfamily 186.2, whereas five J558 subfamilies were utilized in NAAb originating from normal mice. Junctional diversity as a result of N or P nucleotide insertions and D-D fusions was noted among IgMs originating from both mev (mostly B-1 lymphocytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all six J558+ IgMs from mev mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-D-J rearrangements were observed among these IgMs. None of the IgMs were polyreactive and three of the six were subsequently observed to express monospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Three IgM antibodies had hydrophilic arginine residues in their CDR3 heavy chain region. By contrast, all six J558+ IgMs from C57/BL6 mice had variable CDR3 length, distinct VDJ rearrangements and a local negative charge in the CDR3 region. Four of these IgMs demonstrated polyreactivity with multiple conserved autoantigens and, hence, were classified as NAAb. These findings provide evidence for either positive or impaired negative selection of B-1 lymphocytes secreting disease-associated IgM AAb in mev mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction between monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.