The uptake and intracellular distribution of oligonucleotide phosphorothioates (S-oligonucleotides) and their analogs by various cells lines were studied using internally 35S-labeled oligonucleotides. Intracellular accumulation starts in the first 2 hr and reaches a maximum at about 16 hr. A decrease of intracellular concentration of the S-oligonucleotide was observed after 16 hr, probably due to a significant efflux transport. Cellular uptake was dependent on the extracellular concentration. The intracellular concentration was significantly higher than that in the medium. The uptake and the intracellular distribution were different in the various cell lines studied. Comparative studies of the uptake of the S-oligonucleotide and various 3' end-modified S-oligonucleotides show that end modified S-oligonucleotides with a hydrophobic group significantly increases the uptake. The introduction of methylphosphonothioate linkages at the 3' end of the S-oligonucleotide also resulted in an increase in uptake.