Four fluorescent 5-alkynyl-2′-deoxyuridines were studied in DNA oligonucleotides and their duplexes. The fluorescence response to hybridization differs dramatically for nucleosides containing a perylene fluorochrome either conjugated or not conjugated to the nucleobase. The conjugated nucleoside, 5-(perylen-3-ylethynyl)-2′-deoxyuridine, shows enhanced long-wavelength emission in the DNA duplex, in contrast to the blue fluorescence of perylene on a flexible linker (in 5-[(perylen-3-yl)methoxyprop-1-ynyl]-2′- deoxyuridine), which is quenched upon duplex formation. 2006 Wiley-VCH Verlag GmbH & Co. KGaA.