Separate diastereomers of suitably protected dithymidine (3',5')-phenylphosphonates and dithymidine (3',5')-phenylphosphonothioates were obtained via a palladium(0) catalysed stereospecific cross-coupling reaction between separate diastereomers of the corresponding dinucleoside H-phosphonates and dinucleoside H-phosphonothioates with iodobenzene. These compounds were converted into the corresponding phosphoramidite building blocks and used for incorporation of P-stereodefined dithymidine phenylphosphonate and phenylphosphonothioate units TxT into DNA oligonucleotide chain. Dodecathymidylates with centrally positioned one (T(9)TxTT(9)) or two modified units (T(8)TxTTxTT(8)) exhibited different affinity towards complementary DNA (dA(20)) or RNA (A(20)) strands depending on stereochemistry at the phosphorus center, as determined by UV melting temperature studies. Oligonucleotides containing the R-P-phenylphosphonate or R-P -phenylphosphonothioate internucleotide linkages exhibited higher binding affinity to the complementary strands than their S-P-counterparts, but slightly lower than the non-modified reference T-20. All phenylphosphonothioate-modified oligonucleotides formed less stable than their oxo-counterparts duplexes. As expected, the cleavage of the oligonucleotides investigated with 3'- and 5'-exonucleases was stalled at the modification site, independently of P-chirality at the modification site.