Histone H2B is a common target of autoantibodies in both spontaneous and drug-induced systemic lupus erythematosus (SLE). Recent studies demonstrate that Asp(25) of histone H2B (H2B) spontaneously converts to an isoaspartic acid (isoAsp) in vivo. Our laboratory has demonstrated that the posttranslational modification of an aspartic acid to an isoaspartic acid within self-peptides renders otherwise ignored peptides immunogenic. Analysis of serum from lupus-prone mice and histone antibody positive SLE patients revealed antibodies specific to the Asp and isoAsp H2B(21-35) peptide, and that the expression of these antibodies is dependent on TLR9. IsoAsp H2B(21-35) is immunogenic in non-autoimmune prone mice and mice lacking the ability to repair isoAsp have significantly reduced levels of antibodies to H2B. Asp H2B(21-35) incubated at physiological temperatures and pH acquires the isoAsp modification, demonstrating that H2B(21-35) is prone to spontaneous isoAsp formation in vivo. Autoimmunity to isoAsp H2B suggests that this form of the autoantigen may be critical in the induction of anti-histone autoantibodies in human SLE and in murine models of disease.