A novel assay for antibody captured bioactivity (ACB) has been developed to quantitate deoxyribonuclease I (DNase) in human serum samples. The procedure is simple, sensitive, reproducible and has a high throughput. Serum samples are diluted a minimum of 1/4 and assayed in 96-well microtiter plates coated with polyclonal antibodies specific to DNase. The serum is removed from the wells, the plates are washed and the antibody bound DNase is incubated at 37 degrees C with a DNA-methyl green substrate. The assay is sensitive to 0.8 ng/ml with a range to 10 +/- 2 ng/ml, depending upon the time of incubation (48 +/- 2 h). The recovery of rhDNase spiked into human serum samples averaged 84.4% +/- 6.7% in sera diluted 1/4 and 97.8% +/- 7.2% at a 1/8 serum dilution. Intra-assay precision ranged from 3.0 to 7.5% coefficient of variation (% CV) and interassay precision ranged from 5.0 to 10.2% CV for spiked serum controls. Endogenous DNase concentrations in 27 normal human sera were found to range from < 2.0 to 11.4 ng/ml. Endogenous DNase-like activity was found in Cynomolgus and Rhesus monkey sera; this activity diluted linearly and did not interfere with accurate quantitation of added rh DNase. No endogenous DNase-like activity could be detected in ten Sprague-Dawley rat sera. Bovine pancreatic DNase was found to have only very low cross-reactivity in this assay system. The ACB assay format can potentially be applied to the quantitation of other enzymes in serum and other biological samples.