The stimulus for the production of anti-DNA autoantibodies in lupus remains unknown. Since double-stranded DNA (dsDNA) is a weak immunogen, other stimuli such as B cell superantigens or anti-idiotypic antibodies may provide an alternative mechanism for their production. The presence of regulatory determinants on autoantibodies might be revealed through their structural characterization, but they have eluded detection, perhaps because they may be three-dimensional and require closer analysis. In this report we cloned and sequenced the heavy chain variable region (VH) of a monoclonal anti-dsDNA antibody, mAb 3E10, derived from MRL/lpr mice with lupus nephritis previously shown to express an idiotype associated with nephritis in murine and human lupus. We now show that mAb 3E10 VH contains novel structural features unrelated to DNA binding which are shared only by a subset of autoantibodies expressed in murine lupus. These lupus autoantibodies can be distinguished from antibodies of non-autoimmune strains by the presence of a specific sequence at the junction of the diversity and joining genes combined with the use of variable region genes with conserved sequences in framework 1 (FR1) and FR3. The location of the novel sequences indicates the possibility of a three-dimensional solvent-exposed determinant located distant from the classical antigen binding site that could regulate their production, possibly through binding B cell superantigens or other infectious agents.