Antisense phosphorothioate oligodeoxyribonucleotides (PS oligonucleotides) have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. Following administration in vivo, PS oligonucleotides are rapidly cleared from the plasma and distributed to various organs. However, the manner in which administered oligonucleotides are metabolized in plasma and tissues is poorly understood. In this study, a 25-mer PS oligonucleotide (GEM91) complementary to the gag gene mRNA of the human immunodeficiency virus (HIV-1) was administered to mice through intravenous injections to investigate its metabolism. The PS oligonucleotide was extracted from plasma at 1 hour postadministration and from kidney and liver at 24 hours postadministration. After extraction, the PS oligonucleotide and its metabolites were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. The region of interest containing the PS oligonucleotide was then sequenced. Our results show that degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. However, in kidney and liver, degradation was primarily from the 3'-end, but a large proportion of the PS oligonucleotide was degraded from the 5'-end as well. We also studied the metabolism of PS oligonucleotide in plasma after 2-hour intravenous infusion in HIV-infected patients. The degradation of the PS oligonucleotide in plasma was primarily from the 3'-end. This study is important in understanding the metabolism of antisense PS oligonucleotide in vivo in general but also provides guidance for designing second-generation antisense oligonucleotides with improved stability and safety profile.