The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides. S-oligonucleotides had the highest cell binding and uptake, followed by S-O-, O-, and MP-O-oligonucleotides. Competition studies indicated that S-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely block O-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellular O-oligonucleotides was apparent by 4 h, there was no detectable degradation of S-, S-O, or MP-O-oligonucleotides.