Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptormediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.When oligodeoxynucleotides [oligo(dN)s] complementary to the 5' region of c-myc mRNA are added to cells in culture, c-myc protein synthesis is specifically inhibited (1)(2)(3)(4)(5). Furthermore, addition ofantisense oligo(dN)s to cultures inhibits intracellular viral replication (6)(7)(8) Fig. 1 Top depicts a typical fluorescence histogram comparing cells incubated with no oligo(dN) to those incubated for 24 hr with either acridine-labeled oligo(dN) alone or in the presence of excess unlabeled oligo(dN). Intracellular localization of fluorescence was confirmed by fluorescence microscopy of similarly treated cells (Fig. 1 Middle and Bottom). When we examined the rates of accumulation of variously sized acridine-labeled oligo(dN)s we found that the accumulated intracellular fluorescence after incubation of HL60 cells with 12.5 AtM acridine-labeled oligomers [ranging in size from oligo(dT)3 to oligo(dT)20] increased gradually, plateauing within -50 hr after addition of acridine-labeled oligo(dN) to the culture medium ( Fig. 2A). This is in contrast to the 90 min required