Interleukin-8 (IL-8), a monocyte-derived neutrophil chemoattractant factor, is a polymorphonuclear neutrophil chemotaxin that is involved in a number of inflammatory disorders. Transcription of the IL-8 gene is controlled by regulatory proteins, including nuclear factor-κB (NF-κB), a family of proteins that is important in the transcriptional control of a number of genes. When cells are activated, NF-κB translocates from the cytoplasm to the nucleus, where it activates transcription by binding to a specific sequence within the 5′ untranslated region of the gene. During translocation, NF-κB is potentially susceptible to diversion by oligonucleotides that contain the binding sequence for this protein. In the current study, we produced phosphorothioate-modified oligonucleotides containing the specific DNA sequence that NF-κB binds within the IL-8 gene. We then investigated the effects of transfection of monocytes with these oligonucleotides on interleukin-1β (IL-1β)-stimulated IL-8 production, IL-8 mRNA expression, and NF-κB binding activity. We found that transfection with these oligonucleotides significantly inhibited monocyte IL-8 production. A single-stranded oligonucleotide with two copies of the NF-κB-binding sequence was the most potent of those tested. This single-stranded oligonucleotide also inhibited IL-1β-induced translocation of NF-κB to the nucleus and reduced IL-8 mRNA expression. These studies demonstrated that monocyte production of IL-8 can be attenuated using a single-stranded oligonucleotide that binds a transcriptional activating protein before it translocates to the cell nucleus. This approach ultimately may be useful in the control of inflammation involved in a number of diseases.