GTI-2040 is a 20-mer phosphorothioate oligonucleotide, which is complementary to the messenger ribonucleic acid (mRNA) of the R2 subunit of ribonucleotide reductase. This study characterized both the in vivo and in vitro metabolism of GTI-2040. A highly specific ion-pair reversed-phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method was used for the identification of GTI-2040 and metabolites from a variety of biological samples including exonuclease enzyme solutions, plasma, urine, mouse liver/kidney homogenates, and human liver microsomes. Progressively chain-shortened metabolites truncated from the 3' terminal of GTI-2040 were detected in all of the evaluated biological samples. GTI-2040 was found to be a good substrate for 3' but not 5' exonuclease. While the pattern of n-1 chain-shortened 3'-exonucleolytic degradation was similar in the mouse liver and kidney homogenates, the latter was found to contain a larger number of shortenmers, the kidneys appeared to possess higher enzymatic reactivity toward GTI-2040. Thus, metabolism of GTI-2040 was found to occur in a variety of biological samples, mainly mediated by the 3' exonuclease.