Abstract Long-term administration of low doses (5 μg) of recombinant nucleobindin (rNuc), which is an MRL/ lpr/lpr ( MRL l ) mouse-derived DNA-binding protein, induces autoimmunity in both young lupusprone MRL/ + / + ( MRL n ) and normal BALB c mice. In relation to this autoimmunity, using agarose gel electrophoresis we found an approximately 160 bp mono-nucleosomal DNA (nsDNA) in the sera of 6-week-old normal mice 15 h after i.p. injection of 50 μg rNuc. Co-injection of rNuc (50 μg) and anti-CD3 monoclonal antibody (mAb, 50 μg) further accelerated the appearance of nsDNA in the serum together with DNA fragmentation (apoptosis) in the thymus, which had not been so clearly induced by either double amounts of rNuc or anti-CD3 mAb alone. Acceleration of the appearance of nsDNA in the serum by co-injection was also found in age-matched MRL n and MRL l mice, indicating the close association of apoptosis in the thymus with the appearance of nsDNA in the serum. Furthermore, we have detected naturally occurring tri-, di- and mono-nsDNAs from immune complexes (IC) of the sera of 20 ∼ 22-week-old MRL l mice, which indicates that apoptosis in the lymphoid tissues, including the thymus, is the source of serum nsDNA that may trigger or continue production of anti-nuclear antibodies. Evidence that clearance of nsDNA from the circulation is retarded in the presence of rNuc in BALB c mice may give rationale to the induction of autoimmunity in normal mice by long-term administration of even low doses (5 μg) of rNuc after all. Taken together, the unusual appearance of natural Nuc in the circulation may help to trigger and/or maintain the autoimmune status in view of providing nuclear constituents into the immune system through the apoptotic process.