The inhibition of specific transcription regulatory proteins is a new approach to control gene expression. The transcriptional activities of DNA‐binding proteins can be inhibited by the use of double‐stranded oligonucleotides that compete for the binding to their specific target sequences in promoters and enhancers. We used nicked (NDODN‐κB) and circular (CDODN‐κB) dumbbell DNA oligonucleotides containing a NF‐κB binding site to analyze the inhibition of the NF‐κB‐dependent activation of the human immunodeficiency virus type‐1 (HIV‐1) enhancer. The dumbbell DNA oligonucleotides are stable, short segments of double‐stranded DNA with closed nucleotide loops on each end, which confer resistance to exonucleases. The dumbbell and other oligonucleotides (decoys) with the NF‐κB sequence were found to compete with the native strand for NF‐κB binding. The circular dumbbell and double‐stranded phosphorothioate oligonucleotides competed with the native strand for binding to the NF‐κB binding proteins, while the nicked NF‐κB dumbbell was a less effective competitor. In Jurkat T‐cells, the dumbbell and other oligonucleotides were tested for their ability to block the activation of the plasmid HIV‐NL4‐3 Luc. The CDODN‐κB strongly inhibits the specific transcriptional regulatory proteins, as compared with the NDODN‐κB and the double stranded phosphodiester oligonucleotides. On the other hand, the double stranded phosphorothioate oligonucleotides could also block this activation, but the effect was non‐specific. The circular (CDODN) dumbbell oligonucleotides may efficiently compete for the binding of specific transcription factors within cells, thus providing anti‐HIV‐1 or other therapeutic effects.