The Rp- and Sp-diastereomers of the phosphorothioate-containing oligonucleotide d[ApAp(S)ApA] have been synthesized. They and the tetramer d[ApAp(S)ApA] were tested as substrates for staphylococcal nuclease, DNase II and spleen phosphodiesterase. For digestions with DNase I these oligonucleotides were converted to the 5′-phosphorylated derivates. The reactions with the nucleases were analysed by HPLC. The phosphorothioate groups of both diastereomers were resistant to the action of staphylococcal nuclease, DNase I and DNase II. While the phosphorothioate group of the Rp-diastereomer was resistant to the action of spleen phosphodiesterase, the Sp-diastereomer was hydrolysed at an estimated rate 1/100 the rate of cleavage of the unmodified tetramer. The presence of the phosphorothioate group in the center of the molecule affected the rate of hydrolysis of neighbouring phosphate groups for some enzymes. In particular, very slow release of 3′-dAMP from the Rp-diastereomer occurred on incubation with staphylococcal nuclease but the Sp-diastereomer was completely resistant. DNase II produced 3′-dAMP quite rapidly from both diastereomers of d[ApAp(S)ApA] and DNase I released 5′-dAMP from both diastereomers of d[ApAp(S)ApA] only slowly.