Stereo-reproducibility of phosphoroamidite-based phosphorothioate oligonucleotide synthesis is examined through synthesis of singly thioate-substituted oligodeoxynucleotide model compounds. Baseline RP-HPLC resolution of resulting Rp and Sp diastereomers allowed accurate determination of any enantiomeric excess at each phosphorothioate linkage. Our results show that phosphorothioate linkage formation is not a stereo-random process. AH investigated stereoisomeric phosphorothioate diester linkages were formed with a small, reproducible excess of the R isomer (2-6% per linkage). Regardless of the synthesized sequence, all Rp/Sp diastereoisomer ratios were between 50:50 and 60:40, indicating that this synthesis process is under inherent stereochemical control.