The synthesis of oligonucleotides containing 2'-deoxy-2'-fluoro-4'-thioarabinonucleotides is described. 2'-Deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4'S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4'S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4'-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2'-deoxy-2'-fluoro-4'-thioarabinouridine (4'S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2'F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.