Two isomeric dialkynylpyrene phosphoramidites and their incorporation into oligonucleotides are described. The pyrene units closely resemble the well-known perylene bisimide dye PDI with regard to the ability to self-organize within a DNA duplex. In addition, dialkynylpyrenes exhibit significant monomer and remarkably strong excimer fluorescence. The dialkynylpyrene building blocks are promising candidates for applications in diagnostic toots, such as excimer-based molecular beacons, as well as for novel DNA-based materials with special optical properties.