A novel, sensitive colorimetric test is described for quantification of the initial number of hepatitis B virus (HBV) genomes amplified in PCR. The viral genomes are amplified together with a synthetic internal standard (IS) to correct for the variability of the efficiency factor. One of the two primers is biotinylated, and the amplified mixtures of HBV and IS DNAs are bound to streptavidin-coated microtiter plates for quantitative detection. The ratio of HBV to IS DNA is determined for each sample by hybridization with DNP-containing probes and immunoenzymatic detection. The colorimetric detection is quantitative, rapid, and accurate with a dynamic range from approximately 10(8) to > 10(11) DNA molecules. The initial number of HBV genomes in a clinical sample is interpreted from the signal ratio HBV/IS by using a standard curve, obtained from coamplification of known quantities of synthetic HBV templates with IS. The assay quantified 15 viral genomes from 10 microliters of serum, and its dynamic range was up to five orders of magnitude. After the amplification step, the assay takes > 2 hr, and the method is applicable to automation.