Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.