Phosphorothioate modification of internucleoside linkages is widely used to prevent degradation of oligodeoxynucleotide (ODN) therapeutic agents in serum and cells. This modification generally increases ODN potency, but in many instances it is associated with an increase of poorly understood nonspecific effects. In this study, we have found that both cellular retention and nonspecific protein binding are dependent upon the extent of the oligonucleotide's modification. Flow cytometry of cells treated with fluorescein-labeled single-stranded (ss) or double-stranded (ds) ODNs demonstrated that fully phosphorothioate-modified ODNs exhibit much greater cellular association than 3'-terminally modified ODNs (with three 3'-terminal phosphorothioate linkages). Additionally, gel shift assays with either ss- or ds-probes showed that fully phosphorothioate-modified ODNs also exhibit much greater cytoplasmic and nuclear protein binding than either 3'-terminally modified or unmodified ODNs. However, gel shift competition assays showed that transcription factor binding by fully phosphorothioate-modified ds-ODNs was completely nonspecific relative to 3'-terminally modified and unmodified ds-ODNs. These results suggest that the benefits derived from full phosphorothioate modification of ODNs may be negated by increases of nonspecific protein binding and associated sequence-independent effects.