Tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-alpha by anti-TNF-alpha antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-alpha. The efficacies of various siRNA duplexes were tested against TNF-alpha mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-alpha by 55% (p<0.005) with a proportionate reduction in TNF-alpha mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 microg/kg body weight/day for 2 days) of liposome-encapsulated SSL3. The LPS-induced TNF-alpha secretion was inhibited by >60% (p<0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-alpha, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-alpha.