Tumor necrosis factor-α (TNF-α) is an important mediator in sepsis and septic shock. Kupffer cells (KCs) are the resident macrophages of the liver and are potent producers of TNF-α in response to inflammatory stimuli such as bacterial endotoxin or lipopolysaccharide (LPS). Although the effects of exogenous cytokines such as interferon-γ on TNF-α production by macrophages have been fairly well studied, the intracellular pathways regulating KC TNF-α synthesis are largely unknown. We investigated the role of guanylate cyclase and cGMP in LPS-induced KC TNF-α synthesis. Exogenous 8-BrcGMP and dbcGMP increased LPS-stimulated TNF-α synthesis but had no effect on KC TNF-α in the absence of LPS. Sodium nitroprusside (SNP), a nitric oxide-releasing substance that stimulates guanylate cyclase, increased TNF-α synthesis in response to LPS, whereas methylene blue and LY83583, guanylate cyclase inhibitors, decreased KC TNF-α synthesis. The inhibitory effect of methylene blue could be overcome with exogenous dbcGMP or SNP. Our results demonstrate that guanylate cyclase and cGMP mediate LPS-induced KC TNF-α synthesis and suggest that agents that alter cyclic nucleotide metabolism in KCs may affect the response of these cells to inflammation and inflammatory stimuli. J. Leukoc. Biol. 57: 297–302; 1995.