A simple and straightforward synthesis of the pyrimidine 2‘-deoxyribonucleoside cyclic N-acylphosphoramidites RP-1 and SP-1 is described. Specifically, (±)-2-amino-1-phenylethanol 2 was chemoselectively N-acylated to 4 by treatment with ethyl fluoroacetate 3 followed by reaction with hexaethylphosphorus triamide to afford the cyclic N-acylphosphoramidite 5 as a mixture of diastereomeric rotamers (5a and 5b). Condensation of N4-benzoyl-5‘-O-(4,4‘-dimethoxytrityl)-2‘-deoxycytidine 8 with 5 in the presence of 1H-tetrazole gave, after silica gel chromatography, pure RP-1 and SP-1. 31P NMR studies indicated that when RP-1 or SP-1 is reacted with 3‘-O-acetylthymidine and N,N,N‘,N‘-tetramethylguanidine in CD3CN, the dinucleoside phosphotriester SP-9 or RP-9 is formed in near quantitative yield with total P-stereospecificity (δP 144.2 or 143.9 ppm, respectively). Sulfurization of SP-9 or RP-9 generated the P-stereodefined dinucleoside phosphorothioate RP-11 or SP-11 (δP 71.0 or 71.2 ppm, respectively). The 2‘-deoxycytidine cyclic N-acylphosphoramidite derivatives RP-1 and SP-1 were subsequently applied to the solid-phase synthesis of [RP,RP]- and [SP,SP]-trideoxycytidilyl diphosphorothioate d(CPSCPSC), and [RP,SP,RP]-tetradeoxycytidilyl triphosphorothioate d(CPSCPSCPSC). Following deprotection, reversed-phase (RP) HPLC analysis of these oligonucleotide analogues showed a single peak for each oligomer. By comparison, RP-HPLC analysis of purified P-diastereomeric d(CPSCPSC) and d(CPSCPSCPSC) prepared from standard 2-cyanoethyl deoxyribonucleoside phosphoramidites exhibited 4 and 8 peaks, respectively, each peak corresponding to a specific P-diastereomer (see Figure 3A). The thymidine cyclic N-acylphosphoramidite derivatives RP-14 and SP-14 were also prepared, purified, and used successfully in the solid-phase synthesis of [RP]11-d[(TPS)11T]. Thus, the application of deoxyribonucleoside cyclic N-acyl phosphoramidites to P-stereocontrolled synthesis of oligodeoxyribonucleoside phosphorothioates may offer a compelling alternative to the methods currently used for such syntheses.