Antisense oligonucleotides were potentially very powerful tools to modulate gene expression. Progress in chemical modification of oligonucleotides to enhance the strength and stability of interaction, without loosing specificity, has made the antisense strategy very attractive for therapeutic manipulation of the gene expression. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells. In this study we evaluated a new concept for antisense delivery in cellular systems. We have shown that formation of a duplex between the active oligonucleotide (with a chemically modified backbone) and an easily degradable complementary oligodeoxynucleotide in the presence of Lipofectamine 2000 leads to better intracellular uptake and more significant pharmacological effect of the active oligonucleotide. To evaluate our approach we targeted the MDR1 gene, which coded for P-glycoprotein, a membrane ATPase associated with multi-drug resistance in tumor cells. The 2'-O-methyl gapmer antisense RNA (active component of the duplex) was complementary to a site flanking the AUG of the MDR1 message. Effective inhibition of P-glycoprotein expression was attained with sub-micromolar concentrations of duplexes under serum-replete conditions and was much stronger than with traditional single stranded antisense delivery. The results obtained suggested that double stranded delivery could provide a simple and effective means for enhancing cell uptake of pharmacologically active oligonucleotides.