Influence of stereochemistry of the 3'-terminal phosphorothioate (PS)-modified primers was studied in a single base extension (SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo-) 3' --> 5' exonucleolytic activity. The diastereomerically pure PS-labeled primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (R(P) and S(P)). When the SBE reaction was performed in the presence of mispaired 2'-deoxyribonucleoside triphosphates (dNTPs), the "racemic" 3'-phosphorothioate primer mixture resulted in a lower level of 3' --> 5' exonuclease-mediated cleavage products in comparison to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure RP 3'-phosphorothioate primer was examined, the results were largely the same as for the racemic 3'-phosphorothioate primer mixture. In contrast, a 3'-PS primer of S(P) configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3' --> 5' proofreading products, less mispriming, and improved yield of incorporation of the correct nucleotide.