Novel yttrium-90 (90Y)-labeled phosphorothioate antisense oligonucleotides were designed as a potential targeted radionuclide therapeutic agent for malignant tumors. A 15-mer phosphorothioate antisense oligonucleotide, which was complementary to the translation start region of the N-myc oncogene mRNA, was conjugated with isothiocyanobenzyl ethylenediamine tetraacetic acid (SCN-Bn-EDTA), via a C-5-substituted deoxyuridine that had replaced a thymine in the oligonucleotide, and was then labeled with 90Y-acetate. Following purification, the radiochemical purity of the 90-Y-Bn-EDTA-phosphorothioate antisense oligonucleotides was estimated by 2.0% agarose gel electrophoresis, and the specific hybridization of 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide to a phosphorodiester sense oligonucleotide was investigated by 20% polyacrylamide gel electrophoresis in a cell-free system. Radiochemical purity was 98.7 +/- 0.4% at 72 h after labeling and 90.3 +/- 0.9% after 72-h incubation with human normal serum. The 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide hybridized specifically to a complementary phosphorodiester sense oligonucleotide. In conclusion, phosphorothioate antisense oligonucleotides can be labeled stably with 90Y using SCN-Bn-EDTA without loss of hybridization properties.