Dimers of the fluorescent DNA intercalators oxazole yellow and thiazole orange are used for high-sensitivity DNA detection due to their excellent fluorescence properties. Fluorescence lifetime techniques and absorption spectroscopy were used to investigate the DNA binding properties of POPO-1 [4,4,8,8-tetramethyl-4,8- diazaundecamethylene)bis-4-(3-methyl-2,3-dihydrobenzo-1,3-oxazolyl)-2-methylidene] with the double-stranded homopurine-homopyrimidine polynucleotides poly(dA·dT), poly(dG·dC) and calf thymus DNA. The coexistence of different binding modes of POPO-1 with polynucleotides such as bisintercalation and monointercalation was found in connection with minor groove binding as well as electrostatic attachment. At high excess of polynucleotides, bisintercalation is the only existing form of binding whereas an increasing amount of POPO-1 leads to the coexistence of bis- and monointercalated dye molecules. The amount of bound dye increases with decreasing ionic strength of the buffer and is dependent on the polynucleotide itself. The best binding conditions were found with calf thymus DNA, followed by poly(dA·dT) and poly(dG·dC).