It is widely accepted that normal levels of extracellular DNA in the blood are quite low and can be elevated in systemic lupus erythematosus, some cancers, and after irradiation. The goal of this work was the development of a quantitative assay for DNA measurement in serum of healthy donors as well as in patients. Several approaches to IgG antibody induction against native mammalian DNA were tested. Antiserum containing such antibodies was obtained following immunization of rabbits with denatured DNA complexed to methylated BSA (in one of six animals). A time-resolved immunofluorimetric sandwich assay for DNA was developed using the rabbit antibodies. Sensitivity of the assay is 8 pcg/ml and the range 10-100,0000 pcg/ml DNA. The assay was employed for determination of DNA serum content in normals, viral hepatitis patients, and autoimmune thyroiditis patients. Serum DNA concentration in 38 healthy males was 0.62 +/- 0.49 ng/ml (medium +/- SD), as opposed to 2.3 +/- 6.6 [symbol: see text] 105 +/- 175 ng/ml in 19 hepatitis (B and C) patients and 14 autoimmune thyroiditis patients respectively. The high levels of extracellular DNA in circulation may contribute to induction of DNA-hydrolyzing antibodies often found in these disorders. The assay can also be used for measurement of low DNA concentrations against high protein background in other biological fluids.