Members of the Toll-like receptor (TLR) family are essential players in activating the host innate immune response against infectious microorganisms. All TLRs signal through Toll/interleukin 1 receptor domain-containing adapter proteins. MyD88 adapter-like (Mal) is one such adapter that specifically is involved in TLR2 and TLR4 signaling. When overexpressed we have found that Mal undergoes tyrosine phosphorylation. Three possible phospho-accepting tyrosines were identified at positions 86, 106, and 187, and two mutant forms of Mal in which tyrosines 86 and 187 were mutated to phenylalanine acted as dominant negative inhibitors of NF-kappaB activation by lipopolysaccharide (LPS). Activation of THP-1 monocytic cells with the TLR4 agonist LPS and the TLR2 agonist macrophage-activating lipopeptide-2 induced phosphorylation of Mal on tyrosine residues. We found that the Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 could block the endogenous phosphorylation of Mal on tyrosine in cells treated with macrophage-activating lipopeptide-2 or LPS. Furthermore, Btk immunoprecipitated from THP-1 cells activated by LPS could phosphorylate Mal. Our study therefore provides the first demonstration of the key role of Mal phosphorylation on tyrosine during signaling by TLR2 and TLR4 and identifies a novel function for Btk as the kinase involved.