The present study investigated transcriptional inactivation of TNF- gene by nuclear factor binding oligonucleotides (ONs) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specific cis-acting elements necessary for nuclear factor binding and transactivation of TNF- gene by lipopolysaccharide(LPS). LPS inducibility of TNF- was shown to require transcriptional activation by NF-B at multiple binding sites, including the -850 .(1), -655(2), and -510 (3) site, whereas the -210(4) site had no effect. Maximum inducibility was associated with the activation of 3 site. Sequence-specific double-stranded ON targeting this site was most effective in inhibiting TNF- activity induced by LPS. The inhibitory effect of ON on TNF- bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF- production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF- gene can be achieved by using ONs that compete for the nuclear factor binding to TNF- gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases whose etiology is dependent on aberrant gene expression.