The pathways of degradation of oligodeoxynucleotides in plasma from several mammalian species, including human, were investigated. In all cases, hydrolysis occurred exclusively by a 3' to 5' exonucleolytic activity. Human, mouse, and rat plasma degraded oligonucleotides in this fashion at comparable rates, whereas rabbit plasma was severalfold more active. Single-stranded oligonucleotides were more susceptible to hydrolysis than double-stranded oligonucleotides. The rate of hydrolysis was sequence dependent: 3' pyrimidine nucleotides were cleaved more rapidly than 3' purines. The Km and Vmax values for an oligonucleotide 15-mer with the sequence TAGCACCATGGTTTC in human plasma were 50 microM and 4.5 microM/min, respectively. Substitution of the 3'-terminal phosphodiester internucleoside linkage with a phosphotriester rendered this substrate completely resistant to hydrolysis, showing that the enzyme is a pure 3' to 5' exonuclease and that there are no other nucleolytic activities in plasma. Modification at this position is required to inhibit rapid nuclease degradation of antisense compounds in vivo and in tissue culture systems requiring serum.