We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24-bp sequence, centrally located. Biotinylated PCR products from target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe, and the hybrids were determined by using antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a fluorescein-labeled probe, and the hybrids were determined by using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was measured by adding a Ca(2+)-containing light-triggering solution. Alkaline phosphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430 to 315,000 target DNA molecules. Average CVs ranged from 7 to 17%. The proposed system is expected to facilitate the automation and routine use of quantitative PCR.