Abstract DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5′-terminus with fluorescein and the complementary strand was covalently labeled on the 3′-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 n m in 10 min or about 10 p m in 20–30 min. Detection of a 4 p m concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentration down to 20 p m; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 n m. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-μl sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.