Modified uridine derivatives such as 2-thiouridine (s(2)U), pseudouridine (Psi), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base-modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3'-phosphoramidite derivatives of s(2)U and D units (the 3'-phosphoramidite derivative of Psi is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I(2)/pyridine/water which, if applied towards oligonucleotides containing s(2)U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert-butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH(2)/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side-reaction during the routine synthesis of dihydrouridine-containing RNA. Oligonucleotides modified with s(2)U, D, or Psi units are useful models for structure-function studies. Here, the procedure for preparation of siRNA duplexes is described.