A series of novel modifying reagents, including phosphoramidites and solid supports, have been synthesized, and used for the introduction of 1-(phenylethynyl)pyrene (PEPy) and 9,10-bis(phenylethynyl)anthracene (BPEA) fluorescent dyes into predetermined positions of synthetic oligonucleotides. These two fluorophores have been shown to constitute an energy donor-acceptor pair, and can be used as such in fluorescent oligonucleotide probes, designed for the detection and structural studies of nucleic acids. The sensitivity of the probe to duplex formation is demonstrated. The formation of the PEPy and BPEA excimers is reported for the first time on nucleic acids. © Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.