Sera from 20 patients with systemic lupus erythematosus (SLE), selected for elevated titers of antibody to native DNA (nDNA), were examined by indirect immunofluorescence (IF) on tissue culture Hep-2 and rabbit kidney cells. Twelve sera showed a particulate cytoplasmic staining, in addition to nuclear IF. Double IF staining by using a mouse monoclonal anti-nDNA and a human serum containing anti-mitochondrial antibody as probes showed that the cytoplasmic structures recognized by these 12 SLE sera were mitochondria. SLE sera showing mitochondrial staining had high anti-nDNA levels, as assessed by ELISA (3.5 +/- 1.9 O.D.), compared with those not showing this staining pattern (0.8 +/- 0.4 O.D.). Mitochondrial staining was abolished by DNase I pretreatment of the substrates. Liquid phase absorption of serum anti-nDNA with S1 nuclease-treated calf thymus DNA or purified mitochondrial DNA also removed staining. These findings demonstrate that anti-nDNA antibodies from patients with SLE bind to DNA in intact mitochondria. Therefore, mitochondrial IF staining on tissue culture cells in the presence of nuclear staining should be interpreted with caution, because the phenomenon could be entirely related to anti-native DNA. These observations might also provide new insights concerning the nature of immunogenic cellular components stimulating anti-DNA production.