This paper describes an assay for hepatitis B DNA polymerase which is based on the use of phosphonoformic acid (PFA), a known inhibitor of the viral enzyme, with particulate fractions prepared from blood plasma or serum. The assay makes possible the measurement of the viral enzyme activity in the presence of DNA polymerases unrelated to hepatitis B. The activities of the latter were largely but incompletely suppressed in the presence of 0.4 M KCl, and they were altogether excluded from nucleotide incorporation which was inhibited by PFA. Thus, particulate fractions obtained from HBsAg-negative blood had mean DNA polymerase activities of 1 +/- 1 (SD) pmol/liter/hr with a lower 90th percentile range of 0-3 pmol/liter/hr. Particulate fractions prepared from blood that was positive for both HBsAg and HBeAg had polymerase activities of 58 +/- 66 pmol/liter/hr with an upper 90th percentile range of 4-322 pmol/liter/hr. PFA concentration was optimized using a commercial preparation of the inhibitor, in which the degree of purity was determined. The method utilizes 3H-labeled deoxyribonucleoside triphosphates and is performed on 6 ml of plasma or serum. The effects of precursor concentration and specific activities on the rate of nucleotide incorporation were studied, and the optimal combinations were indicated. Other parameters of the proposed assay were also studied.