The d-allo-and l-talo-hept-6-ynofuranosyluracil-derived phosphoramidites 11A and 11T were prepared in 9 ± 10% yield over eight steps from the previously described propargylic alcohols 1A and 1T, respectively. The corresponding nucleotides were incorporated into rU 14 by standard solid-phase synthesis. While the duplex consisting of rU 14 with one l-talo-hept-6-ynofuranosyluracil in the middle of the strand and rA 14 (I ´V) had the same melting point as the reference duplex rU 14 ´rA 14 (I ´II), the duplex with one d-allo-hept-6-ynofuranosyluracil in the middle of rU 14 and rA 14 (I ´III) melted 1.58 lower than the reference duplex. The duplex I ´VI consisting of rU 14 with six l-talo-hept-6-ynofuranosyluracils distributed over the entire strand and rA 14 showed a melting point that is 118 lower than the reference duplex. The corresponding duplex I ´IV of rU 14 possessing six d-allo-hept-6-ynofuranosyluracils and rA 14 showed a melting point which is more than 208 below the one of the reference duplex. These results are in qualitative agreement with the predictions based on the conformational analysis of the nucleosides and the interference of the ethynyl moiety with the hydration of the oligonucleotides.