Technical Abstract: Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases are known to be activated by LPS in monocytes and macrophages. Therefore, we determined the role of Src tyrosine kinases in TLR4 signaling pathways and target gene expression in macrophages. LPS-induced activation of NF'B and p38 MAPK and expression of inducible nitric oxide synthase (iNOS) were not affected in macrophages deficient in three Src kinases (Lyn, Hck, and Fgr). These results suggest that the deletion of these three Src kinases is not sufficient to abolish Src kinase activities possibly due to the functional redundancy of other Src kinases present in macrophages. However, two structurally unrelated pan-inhibitors of Src kinases, PP1 and SU6656, suppressed LPS-induced iNOS expression in both wild-type and MyD88-deficient macrophages. The suppression of iNOS expression by the inhibitor was correlated with downregulation of IFNß expression and a subsequent decrease in STAT1 phosphorylation. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNß suggesting that Src kinases are involved in primary downstream signaling pathways of TLR4, but not secondary signaling pathways downstream of IFNß receptor. Moreover, PP1 suppressed TRIF-induced expression of IFNß and iNOS. Together, these results demonstrate that Src tyrosine kinases play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of Src-family tyrosine kinases in TLR4 signaling pathways and inflammatory target gene expression.