For the enzymatic digestion of a 25-mer phosphorothioate (PS) oligonucleotide, the reaction kinetics was previously determined to be the sum of two parallel processes: a fast and a very slow phase of digestion suggesting a two-exponential model. A characteristic metabolite profile was observed both in vitro and in vivo. This behavior is shown to be the result of the stereoselective cleavage of chiral R-configuration and S-configuration PS internucleotide linkages by 3'-exonucleases. The stereoselective nature of 3'-exonuclease action was analyzed by reverse-phase HPLC. The separation of eight diastereomers of the tetramer TTCT (5'-3') was used to follow the stereoselective course of exonuclease hydrolysis of PS internucleotide linkages. Degradation of the 25-mer parent compound having a 3' S-terminal internucleotide linkage was calculated to be more than 300 times slower than an analog with a 3'-terminal R-configuration. These results support an approach for protecting antisense oligonucleotides based on the chirality of only the 3'-end internucleotide linkage.