Hybridization of 20mer probe oligonucleotides to complementary, surface-immobilized target oligonucleotides wasvisualized on a single-molecule basis by fluorescencemicroscopy. Coincident determination of the positions ofboth the target and the probe oligonucleotides using dual-wavelength fluorescence labeling allowed for highly reliable discrimination of specifically bound probe moleculesfrom those being physisorbed. The figures of merit of theassay are characterized by the low probability for falsepositive (10-4) events and the high speed for detection ofup to hundreds of different DNA fragments per second.The probability for false negative events is limited by thebiochemical binding probability of short oligonucleotides.The potentials and limitations of this methodology forsingle-cell single-DNA analysis are discussed.